Non-HLA restricted T cell receptors and uses thereof

Abstract

NON-HLA RESTRICTED T CELL RECEPTORS AND USES THEREOF Abstract: The presently disclosed subject matter provides methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to novel designs of T cell receptors (TCRs) and engineered immunoresponsive cells comprising the same. The novel TCR binds to an antigen in an HLA-independent manner. In certain embodiments, the novel TCR provides enhanced sensitivity for a target gene having a low expression level. NON-HLA RESTRICTED T CELL RECEPTORS AND USES THEREOF Abstract: The presently disclosed subject matter provides methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to novel designs of T cell receptors (TCRs) and engineered immunoresponsive cells comprising the same. The novel TCR binds to an antigen in an HLA-independent manner. In certain embodiments, the novel TCR provides enhanced sensitivity for a target gene having a low expression level. 20 24 22 01 00 26 S ep 2 02 4 N O N - H L A R E S T R I C T E D T C E L L R E C E P T O R S A N D U S E S T H E R E O F 2 0 2 4 2 2 0 1 0 0 2 6 2 0 2 4 S e p A b s t r a c t :

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of Australia Patent Application No. 2019216982, which is the national phase entry of International PCT Patent Application No. PCT / US2019 / 017525, which claims priority to US provisional patent application No. 62 / 629,072, filed February 11, 2018, the content of each of which is incorporated by reference in its entirety, and to which priority is claimed. REFERENCE TO A SEQUENCE LISTING Preceding applications contained a Sequence Listing which was originally submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy is named "0727340845SL.txt" and is 71 kilobytes in size. The present application contains a sequence listing which has been submitted electronically as an XML document in the ST.26 format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 25, 2024, is named "P0024173AUD1 - Sequence Listing,xml" and is 74 kilobytes in size. INTRODUCTION The presently disclosed subject matter provides methods and compositions for enhancing the immune response toward cancers and pathogens. It relates to novel designs of T cell receptors (TCRs) and engineered immunoresponsive cells comprising the same. The engineered immunoresponsive cells comprising the novel TCRs are antigen-directed. BACKGROUND OF THE INVENTION Adoptive immunotherapy using antigen recognizing receptors (e.g., chimeric antigen receptors (CARs)) has shown remarkable clinical results in the treatment of leukemia and is one of the most promising new strategies to treat cancer. To generate CAR therapies, current clinical protocols employ autologous T cells and randomly integrating vectors, including gamma-retroviral, lentiviral and transposons, which all result in semi-random integration and variable expression of the CAR owing to transgene variegation. Altogether, the conjunction of autologous cell sourcing and random vector integration is prone to generating cell products with variable potency. Thus, there is a need of novel designs of antigen recognizing receptors having consistent potency and increased ability to detect low levels of target antigen. 2024220100   26 Sep 2024 SUMMA RY OF THE INVENTION The presently disclosed subject matter generally provides HLA-independent (or non-HLA restricted) T cell receptors (referred to as “HI-TCRs”) that bind to an antigen of interest in an HLA-independent manner and immunoresponsive cells comprising thereof. The presently disclosed subject matter also provides methods of using such cells for inducing and / or enhancing an immune response to a target antigen, and / or treating and / or preventing neoplasia or other diseases / disorders where an increase in an antigen-specific immune response is desired. 2024220100   26 Sep 2024 The presently disclosed subject matter provides recombinant T cell receptor (TCR) comprising an antigen binding chain that comprises an extracellular antigenbinding domain and a constant domain, wherein the recombinant TCR binds to an antigen in an HLA-independent manner. 5          In certain embodiments, the constant domain comprises a native or modified TRAC peptide, and / or a native or modified TRBC peptide. In certain embodiments, the constant domain is capable of forming a homodimer or a heterodimer with another constant domain. In certain embodiments, the recombinant TCR is expressed from an expression 0 cassette placed in an endogenous TRAC locus and / or a TRBC locus of an immunoresponsive cell. In certain embodiments, the placement of the recombinant TCR expression cassette disrupts or abolishes the endogenous expression of a TCR comprising a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell. In certain embodiments, the placement of the recombinant TCR expression cassette prevents 15 or eliminates mispairing between the recombinant TCR and a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell.In certain embodiments, the antigen binding chain is capable of associating with a CD3(^ polypeptide. The antigen binding chain, upon binding to an antigen, is capable of activating the CD3(^ polypeptide associated to the antigen binding chain. The activation of the CD3(^ polypeptide is 20 capable of activating an immunoresponsive cell. The CD3(^ polypeptide can be endogenous or exogenous. In certain embodiments, the CD3(^ polypeptide is endogenous and is endogenous and integrated in the native CD3 complex. In certain embodiments, the CD3(^ polypeptide is exogenous and optionally integrated with a co-stimulatory molecule selected from the group consisting of a CD28 polypeptide, a 4-IBB polypeptide, an 25 OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide and any combination thereof. In certain embodiments, the antigen binding chain further comprises a costimulatory region, wherein the recombinant TCR, upon binding to an antigen, is capable of stimulating an immunoresponsive cell. The co-stimulatory region can include a costimulatory molecule selected from the group consisting of a CD28 polypeptide, a 4-1BB 30 polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide and any combination thereof. In certain embodiments, the co-stimulatory region comprises a CD28 polypeptide. In certain embodiments, the recombinant TCR is capable of associating with a CD3 complex. In certain embodiments, the recombinant TCR is capable of integrating 2024220100   26 Sep 2024 with a CD3 complex and providing HLA-independent antigen recognition. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the recombinant TCR replaces an endogenous TCR in a CD3 / TCR complex. In certain embodiments, the extracellular antigen-binding domain is capable of 5 dimerizing with another extracellular antigen-binding domain. The extracellular antigenbinding domain can include a ligand for a cell-surface receptor, a receptor for a cell surface ligand, an antigen binding portion of an antibody or a fragment thereof or an antigen binding portion of a TCR. In certain embodiments, the extracellular antigenbinding domain comprises a heavy chain variable region (Vh) of an antibody, or a VHH 10 from a camelid Vu-only antibody and / or a light chain variable region (Vl) of an antibody. In certain embodiments, the extracellular antigen-binding domain is capable of dimerizing with another extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a Vh of an antibody, wherein the human, murine, or camelid Vh is capable of dimerizing with another extracellular 15   antigen-binding domain comprising a Vl of the antibody and form a fragment variable (Fv). In certain embodiments, the extracellular antigen-binding domain comprises a Vl of an antibody, wherein the Vl is capable of dimerizing with another extracellular antigen-binding domain comprising a Vh of the antibody and form a fragment variable (Fv). 20          In certain embodiments, the recombinant TCR binds to a tumor antigen. The tumor antigen can be selected from the group consisting of CD 19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R- 25   a2, K-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, Mesothelin, ERBB2, MAGEA3, p53, MARTI,GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, and CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB. In 30 certain embodiments, the tumor antigen is CD19. In certain embodiments, the recombinant TCR exhibits greater antigen sensitivity than a CAR targeting the same antigen. In certain embodiments, the recombinant TCR is capable of inducing an immune response when binds to an antigen that has a low density 2024220100   26 Sep 2024 on the surface of a tumor cell. In certain embodiments, the antigen that has a low density on the cell surface has below about 2,000 molecules per cell. The presently disclosed subject matter further provides immunoresponsive cells comprising a recombinant TCR described herein. In certain embodiments, the expression 5 cassette of at least one antigen binding chain of the recombinant TCR is placed at an endogenous gene locus of the immunoresponsive cell. In certain embodiments, the expression cassettes of two antigen binding chains of the recombinant TCR are placed at an endogenous gene locus of the immunoresponsive cell, wherein the two antigen binding chains are capable of dimerization. The placement of the expression cassette of the 0 recombinant TCR can disrupt or abolish the endogenous expression of a TCR comprising a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell, whereby preventing or eliminating mispairing between the recombinant TCR and a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell. The endogenous gene locus can be a CD38 locus, a CD3e locus, a CD247 locus, &B2Mlocus, a TRAC locus, a 15 TRBC locus or a TRGC locus or a TRBC locus. In certain embodiments, the endogenous gene locus is a TRAC locus and / or a TRBC locus. The endogenous gene locus can include a modified transcription terminator region. In certain embodiments, the modified transcription terminator region comprises a genomic element selected from the group consisting of a TK transcription terminator, a GCSF transcription terminator, a TCRA 20 transcription terminator, an HBB transcription terminator, a bovine growth hormone transcription terminator, a SV40 transcription terminator and a P2A element; the P2A element allows the use of the endogenous transcription terminator of the targeted gene. In certain embodiments, when one endogenous T cell receptor locus in a cell is modified to express the at least one antigen binding chain of the recombinant TCR, one or more 25 other endogenous T cell receptor locus in the cell is modified to eliminate the expression of an endogenous TCR chain. In certain embodiments, the one or more other endogenous T cell receptor loci are further modified to express a gene of interest. The gene of interest can be an anti-tumor cytokine, a co-stimulatory molecule ligand, a tracking gene or a suicide gene. In certain embodiments, one or more endogenous TCR loci are further 30 modified to incorporate a sequence encoding co-stimulatory signaling domain(s) to generate a TCR chain containing such signaling domain(s) at the carboxy terminus. In certain embodiments, the immunoresponsive cell is selected from the group consisting of a T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a human embryonic stem cell, and a pluripotent stem cell from 2024220100   26 Sep 2024 which lymphoid cells may be differentiated. In certain embodiments, the immunoresponsive cell is autologous. In certain embodiments, the immunoresponsive cell further comprises at least one exogenous co-stimulatory ligand. In certain embodiments, the co-stimulatory ligand is 5 selected from the group consisting of CD80, CD86, 41BBL, CD275, CD40L, OX40L and any combination thereof. In certain embodiments, the cell further comprises or consists of one exogenous co-stimulatory ligand. In certain embodiments, the one exogenous costimulatory ligand is CD80 or 4-1BBL. In certain embodiments, the cell further comprises or consists of two exogenous co-stimulatory ligands. In certain embodiments, 10 In certain embodiments, the two exogenous co-stimulatory ligands are CD80 and 4-1BBL. In certain embodiments, the immunoresponsive cell further comprises at least one chimeric costimulatory receptor (CCR). In certain embodiments, the CCR comprising a co-stimulatory molecule selected from the group consisting of a CD28 polypeptide, a 415 IBB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide and any combination thereof. The presently disclosed subject matter also provides pharmaceutical compositions comprising the immunoresponsive cell(s) disclosed herein and a pharmaceutically acceptable excipient. The pharmaceutical composition can be used for treating a 20 neoplasia. Further provided are methods of reducing tumor burden in a subject. In addition, the presently disclosed subject matter providesmethods of lengthening survival of a subject having a neoplasm (e.g., cancer). In certain embodiments, the method comprises administering to the subject an effective amount of the immunoresponsive cells described 25 herein or the pharmaceutical composition described herein. The presently disclosed subject matter also provides methods of treating or preventing a neoplasm. In certain embodiments, the method comprises administering to the subject an effective amount of the immunoresponsive cells described herein or the pharmaceutical composition described herein. The neoplasm can be selected from the 30   group consisting of blood cancer, B cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, non-Hodgkin’s lymphoma and adenocarcinoma. In certain embodiments, the neoplasm is B cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, or non-Hodgkin’s lymphoma, and the recombinant TCR binds to CD 19. In 2024220100   26 Sep 2024 certain embodiments, the neoplasm is B cell leukemia, multiple myeloma, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia, an adenocarcinoma, or non-Hodgkin’s lymphoma, and the recombinant TCR binds to CD 19, MUC16, MUC1, CA1X, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, 5   CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, LI cell adhesion molecule, MAGE-A1, Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI,GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, NY-ES0-1, oncofetal antigen (h5T4), PSCA, 0 PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB. In certain embodiments, the neoplasm is CD 19+ ALL. The presently disclosed subject matter further provides methods for producing an antigen-specific immunoresponsive cell. In certain embodiments, the method comprises 15 introducing into an immunoresponsive cell a nucleic acid sequence encoding a recombinant TCR described herein. The nucleic acid sequence can be comprised in a vector. In certain embodiments, the expression cassette of at least one antigen binding chain of the recombinant TCR is placed at an endogenous gene locus of the immunoresponsive cell. In certain embodiments, the expression cassettes of two antigen 20   binding chains of the recombinant TCR are placed at an endogenous gene locus of the immunoresponsive cell, wherein the two antigen binding chains are capable of dimerization. The endogenous gene locus can be a CD38 locus, a CD3e locus, a CD247locus, a B2M locus, a TRAC locus, a TRBC locus, a TRDC locus and / or a TRGC locus. In certain embodiments, the endogenous gene locus is a TRAC locus or a TRBC 25 locus. In certain embodiments, the placement of the expression cassette of the recombinant TCR disrupts or abolishes the endogenous expression of a TCR comprising a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell, whereby preventing or eliminating mispairing between the recombinant TCR and a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell. In certain 30 embodiments, the endogenous gene locus comprises a modified transcription terminator region. In certain embodiments, the modified transcription terminator region comprises a genomic element selected from the group consisting of a TK transcription terminator, a GCSF transcription terminator, a TCRA transcription terminator, an HBB transcription terminator, a bovine growth hormone transcription terminator, an SV40 transcription 2024220100   26 Sep 2024 terminator and a P2A element. In certain embodiments, when one endogenous T cell receptor locus in a cell is modified to express the at least one antigen binding chain of the recombinant TCR, one or more other endogenous T cell receptor locus in the cell is modified to eliminate the expression of an endogenous TCR chain. In certain 5 embodiments, the one or more other endogenous T cell receptor locus are further modified to express a gene of interest. In certain embodiments, the gene of interest is an anti-tumor cytokine, a co-stimulatory molecule ligand, a tracking gene or a suicide gene. The presently disclosed subject matter further provides nucleotide acids encoding a recombinant TCR described herein, and nucleic acid compositions comprising a 10 recombinant TCR described herein. In certain embodiments, the nucleic acid sequences are comprised in a vector. The presently disclosed subject matter also provides vectors comprising the nucleic acid composition described herein. Further provided are kits comprising a recombinant TCR described herein, an immunoresponsive cell described herein, a pharmaceutical composition described herein, a nucleic acid composition 15 described herein, or a vector described herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm, a pathogen infection, an autoimmune disorder, or an allogeneic transplant. Ilustrative neoplasms for which the presently disclosed subject matter can be used include, but are not limited to leukemias (e.g., acute leukemia, acute lymphocytic 20 leukemia, acute myelocytic leukemia a, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin’s disease, non-Hodgkin’s disease), Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors such as 25 sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, 30 adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder 2024220100   17 Sep 2026 carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). The presently disclosed subject matter further provides use of any recombinant TCR, 5 any pharmaceutical composition or any immunoresponsive cell disclosed herein for use in a therapy. The presently disclosed subject matter further provides a method of treating a tumor cell in a subject, the method comprising administering to the subject an effective amount of immunoresponsive cells, wherein the immunoresponsive cell comprises a recombinant T cell 0 receptor (TCR) that comprises: i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; and ii) a second antigenbinding chain comprising an antigen-binding fragment of a light chain variable region (VL) of the antibody; wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind 15 to an antigen, and wherein the tumor cell expresses the antigen on its surface at a density of less than about 10,000 molecules per cell. The presently disclosed subject matter further provides a use of an immunoresponsive cell for the manufacture of a medicament for treating a tumor cell in a subject, wherein the immunoresponsive cell comprises a recombinant T cell (TCR) that comprises: i) a first 20 antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; and ii) a second antigen-binding chain comprising an antigenbinding fragment of a light chain variable region (VL) of the antibody; wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind to an antigen, and wherein the tumor 25 cell expresses the antigen on its surface at a density of less than about 10,000 molecules per cell. The presently disclosed subject matter further provides a method of treating a tumor cell in a subject, the method comprising administering to the subject an effective amount of immunoresponsive cells, wherein the immunoresponsive cell comprises a recombinant T cell 30 receptor (TCR) that comprises: i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; and ii) a second antigenbinding chain comprising an antigen-binding fragment of a light chain variable region (VL) of the antibody; wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind 2024220100   17 Sep 2026 to an antigen with a dissociation constant (Kd) of about 1x10-8 M or less, and the tumor cell expresses the antigen. The presently disclosed subject matter further provides a use of an immunoresponsive cell for the manufacture of a medicament for treating a tumor cell in a subject, wherein the immunoresponsive cell comprises a recombinant T cell (TCR) that comprises: i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; and ii) a second antigen-binding chain comprising an antigenbinding fragment of a light chain variable region (VL) of the antibody; wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind to an antigen with a dissociation constant (KD) of about 1x10-8 M or less, and the tumor cell expresses the antigen. 2024220100   17 Sep 2026 BRIEF DESCRIPTION OF THE FIGURES The following Detailed Description, given by way of example, but not intended to limit the presently disclosed subject matter to specific embodiments described, may be understood in conjunction with the accompanying drawings. 5         Figures 1A-1E depict HLA-Independent TCR-based Chimeric Antigen Receptor HIT (HIT-CAR, i.e., HI-TCR or HIT) and gene targeting strategy at the TRAC locus in human T cells. (A) Schematic representation of the T Cell Receptor (TCR), the B Cell Receptor (BCR), a Chimeric Antigen Receptor (CAR) and the HLA-Independent TCR-based Chimeric Antigen Receptor (HIT-CAR, i.e., HI-TCR or HIT). (B) CRISPR / Cas9-targeted integration of 0 the 3 receptors into the TRAC locus. Top: TRAC locus; middle: rAAV6 containing the different receptor cassette flanked by homology arms. (C) Representative TCR / Mouse F(ab')2 flow cytometry plots 4 days after TRAC targeting. The TCR antibody epitope recognize the constant chain of the TCR alpha and beta. (D) Cytotoxic activity using an 18-hour bioluminescence assay, using firefly luciferase (FFL)-expressing NALM-6 as targets cells 15   (n=3). (E) Relative CAR MFI (1=MFI at Oh) of CAR T cells after 1, 2 or 4 (arrows) stimulations on CD19 positive target cells. Figures 2A-2B depict expression and therapeutic efficacy of HI-TCR. (A) Representative TCR / Mouse F(ab')2 flow cytometry plots 4 days after TRAC targeting. (B) Kaplan-Meier analysis of the mice survival where NALM-6- bearing mice were treated with 20   5x105 CAR T cells. Figures 3A-3E depict gene targeting strategy and expression of NYESO TCR. (A) Schematic representation of the NYESO TCR genes integrated into the TCR alpha or beta chain. (B) Representative TCR-V-beta-1 flow cytometry plots 4 days after TRAC or TRBC targeting. (C) Cytotoxic activity using an 18-hour bioluminescence assay, using firefly 25 luciferase (FFL)-expressing PC3 as targets cells (n=3). (D) Schematic representation of a cotargeting into both the TCR alpha and the TCR beta. (E) 2024220100   26 Sep 2024 Representative TCR-V-beta-l / 4-lBBL flow cytometry plots 4 days after TRAC and TRBC co- targeting. Figures 4A-4C depict strategy of integrating a CAR into a TRAC locus and modulation the expression by various transcriptional termination signals / 3' unstranslated 5 regions (3’UTR). (A) Schematic representation of the 1928z CAR gene integrated into the TRAC locus. Poly A (black box) corresponds to the segment of the CAR cassette that was modified to test different viral and mammalian 3' UTRs. (B) Representative CAR flow plots 3 days after TRAC (left panel).and geometric mean fluorescence intensity (gMFI), MFI, and Median values for the CAR-expressing population (right panel). Boxed 10 is the original 3'UTR sequence of bovine growth hormone poly A. (C) Absolute (top) and relative (bottom) CAR MFI (1=MFI at Oh) of CAR T cells after 1, 2 stimulations on CD19 positive target cells (as shown in Fig. 1). Figures 5A and 5B depict efficacy of genetically integrated CARs with different 3’UTR sequences. (A) FFL-NALM-6-bearing mice were treated with 1 * 105 CAR T 15 cells, where tumor burden is shown as bioluminescent signal quantified per animal 14 days post T-cell injection, n = 6 mice per group. (B) Tumor burden (average radiance) of NALM-6-bearing mice treated with 1 * 105 CAR T cells (n = 6; line = one mouse) quantified at days 7, 14, and 21 post T-cell injection. Figures 6A-6C depict HIT gene targeting at the TRAC locus in human T cells. (A) 20 CRISPR / Cas9-targeted CAR or HIT gene integration into the TRAC locus. Top, TRAC locus; middle, rAAV6 containing the CAR cassette flanked by homology arms; bottom, rAAV6 containing the HIT cassette flanked by homology arms. (B) Representative CAR / HIT flow plots 4 days after transfection of T cells with Cas9 mRNA and TRAC gRNA and addition of AAV6. CAR and HIT surface protein were detected using a goat 25 anti-mouse IgG. (C) Average CAR / HIT mean fluorescence intensity (MFI) analyzed by FACS 4 days after transduction (n = 6 independent experiments). Figures 7A-7B depict HIT T cells outperform CAR T cells at killing target cells expressing low antigen levels. Nalm6 cell line (expressing firefly luciferase) was gene edited at the CD 19 locus using CRISPR / Cas9, and clones expressing different CD 19 30 levels were generated. (A) FACS analysis of representative Nalm6 clones for each CD19 expression level group (Neg = negative). (B) Cytotoxic activity using an 4h bioluminescence assay, using NALM6 as targets cells expressing different CD 19 levels and CAR (red squares) or HIT (blue circles) T cells at 1:1 effector (E): target (T) ratio. 2024220100   26 Sep 2024 Figures 8A-8C depict HIT T cells outperform CAR T cells at killing target cells expressing low antigen levels. Cytotoxic activity using an 18 h bioluminescence assay, using NALM6 as targets cells expressing different CD 19 levels (indicated at the right), which were incubated with untransduced T cells (A), CAR T cells (B), and HIT T cells 5    (C) at different effector (E): target (T) ratios. Figures 9A-9D depict HIT T cells expressing costimulatory ligands outperform CAR T cells in controlling established B-ALL tumor with very low CD 19 levels. NALM-6-bearing mice were treated with 4x105 untransduced (NT), CAR, or HIT T cells. Tumor burdeAsEPjwas quantified weekly over a 54-day period using BLI. Quantification is the 0   average photon count of ventral and dorsal acquisitions per animal at all given time points. Each line represents one mouse, and n = 5 mice per group. (A) Untransduced (black) vs CAR (red) T cells. (B) CAR (red) vs HIT (green) T cells. (C) T cells expressing HIT alone (green), HIT + CD80 co-stimulatory ligand (orange), HIT + 41BBL co-stimulatory ligand (pink), or HIT + CD80 + 41BBL co-stimulatory ligands (blue). (D) 15 Mouse survival analysis. Figures 10A-10C depict that baseline TRAC-CAR expression can be controlled by distinct 3'UTR sequences without affecting cell surface replenishment kinetic after antigen encounter. (A) CRISPR / Cas9-targeted CAR gene integration into the TRAC locus. The targeting construct (AAV) contains the 1928z CAR coding sequence followed 20 by a 3'UTR sequence, flanked by sequences homologous to the TRAC locus (LHAjsEpjand RHA, left and right homology arm). (B) Each 3'UTR sequence provides different CAR surface levels (measured by FACS). TK thymidime kinase (short version); GCSF: human GCSF, derived from pEF-BOS plasmid; TCRa: TCR alpha, exon 4; HBB: human B-globin; RBG: rabbit B-globin; SV40: simian virus 40 poly A; P2A: porcine teschovirus-1 25 self-cleaving 2A sequence; it allowed the use of the endogenous TRAC poly A sequence. (C) CAR T cells were stimulated once (indicated by a red arrow) with CD19-expressing 3T3 cells, and CAR MFI was measured every 24h over a 3-day period. All CAR T cells show similar CAR expression regulation. 30          DETAILED DESCRIPTION OF THE INVENTION The presently disclosed subject matter provides HLA-independent (or non-HLA restricted) T cell receptors (referred to as “HI-TCRs”) that bind to an antigen of interest in an HLA-independent manner. In certain embodiments, the HI-TCRs are TCR molecules where the TCR variable domain is replaced by the variable domain from an antibody 2024220100   26 Sep 2024 (Fv), resulting in a FvTCR. In certain non-limiting embodiments, the HI-TCR can bind to a tumor antigen or a pathogen antigen. The presently disclosed subject matter also provides cells, including genetically modified immunoresponsive cells (e.g., T cells, NKT cells, or CTL cells) comprising the presently disclosed HI-TCR. In certain non-limiting 5 embodiments, binding of the antigen by the HI-TCR is capable of activating the immunoresponsive cell. The presently disclosed subject matter also provides methods of using such cells for inducing and / or enhancing an immune response to a target antigen, and / or treating and / or preventing neoplasia or other diseases / disorders where an increase in an antigen-specific immune response is desired. 10   1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art. The following references provide one of skill with a general definition of many of the terms used in the presently disclosed subject matter: Singleton et al., Dictionary of Microbiology and Molecular 15 Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. 20          As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art Alternatively, “about” can mean a range of up to 25 about 20%, e.g., up to about 10%, up to about 5%, or up to about 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within about 5-fold or within about 2-fold, of a value. By “activates an immunoresponsive cell” is meant induction of signal transduction 30 or changes in protein expression in the cell resulting in initiation of an immune response. For example, when CD3 Chains cluster in response to ligand binding and immunoreceptor tyrosine-based inhibition motifs (ITAMs) a signal transduction cascade is produced. In certain embodiments, when an endogenous TCR or an exogenous CAR binds to an antigen, a formation of an immunological synapse occurs that includes 2024220100   26 Sep 2024 clustering of many molecules near the bound receptor (e.g. CD4 or CD8, CD3y / 6 / s / ^, etc.). This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation in turn initiates a T cell activation pathway ultimately activating transcription factors, such as 5 NF-kB and AP-1. These transcription factors induce global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response. By “stimulates an immunoresponsive cell” is meant a signal that results in a robust and sustained immune response. In various embodiments, this occurs after 0 immune cell (e.g., T-cell) activation or concomitantly mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40 and ICOS. Receiving multiple stimulatory signals can be important to mount a robust and long-term T cell mediated immune response. T cells can quickly become inhibited and unresponsive to antigen. While the effects of these co-stimulatory signals may vary, they generally result 15 in increased gene expression in order to generate long lived, proliferative, and anti-apoptotic T cells that robustly respond to antigen for complete and sustained eradication. The term “antigen recognizing receptor” as used herein refers to a receptor that is capable of activating an immune or immunoresponsive cell (e.g., a T-cell) in response to its binding to an antigen. Non-limiting examples of antigen recognizing receptors include 20 native or endogenous T cell receptors (“TCRs”), and chimeric antigen receptors (“CARs”). As used herein, the term “antibody” means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and 25 in vivo. Accordingly, as used herein, the term “antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab. F(ab')2, and Fab fragments that lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. NucL Med. 24:316-325 (1983). As used herein, antibodies include whole 30 native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies. In certain embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised 2024220100   26 Sep 2024 of a heavy chain variable region (abbreviated herein as Vh) and a heavy chain constant (Ch) region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as Vl) and a light chain constant Cl region. The light chain constant region is 5 comprised of one domain, Cl. The Vh and Vl regions can be further sub-divided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each Vh and VlIs composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. 10 The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system. As used herein, “CDRs” are defined as the complementarity determining region 15 amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th U. S. Department of Health and Human Services, National Institutes of Health (1987). Generally, antibodies comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of 20 contact residues for the binding of the antibody to the antigen or epitope. In certain embodiments, the CDRs regions are delineated using the Kabat system (Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). As used herein, the term “single-chain variable fragment” or “scFv” is a fusion 25 protein of the variable regions of the heavy (Vh) and light chains (Vl) of an immunoglobulin covalently linked to form a Vh::Vl heterodimer. The Vh and Vl are either joined directly or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), which connects the N-terminus of the Vh with the C-terminus of the Vl, or the C-terminus of the Vh with the N-terminus of the Vl. The linker is usually rich in glycine for 30 flexibility, as well as serine or threonine for solubility. Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid including Vh - and Vl -encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 2024220100   26 Sep 2024 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol2009 183(4):2277-85; Giomarelli et al., Thromb 5 Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunoll997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 0   2003 1638(3):257-66). As used herein, the term “affinity” is meant a measure of binding strength. Affinity can depend on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, and / or on the distribution of charged and hydrophobic groups. As used herein, the term “affinity” 15 also includes “avidity”, which refers to the strength of the antigen-antibody bond after formation of reversible complexes. Methods for calculating the affinity of an antibody for an antigen are known in the art, including, but not limited to, various antigen-binding experiments, e.g., functional assays (e.g., flow cytometry assay). The term “chimeric antigen receptor” or “CAR” as used herein refers to a 20 molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signaling domain that is capable of activating or stimulating an immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises a scFv. The scFv can be derived from fusing the variable heavy and light regions of an antibody. Alternatively or 25 additionally, the scFv may be derived from Fab’s (instead of from an antibody, e.g., obtained from Fab libraries). In certain embodiments, the scFv is fused to the transmembrane domain and then to the intracellular signaling domain. In certain embodiments, the CAR has a high binding affinity or avidity for the antigen. As used herein, the term “nucleic acid molecules” include any nucleic acid 30 molecule that encodes a polypeptide of interest or a fragment thereof. Such nucleic acid molecules need not be 100% homologous or identical with an endogenous nucleic acid sequence, but may exhibit substantial identity. Polynucleotides having “substantial identity” or “substantial homology” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By 2024220100   26 Sep 2024 “hybridize” is meant a pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). 5           For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, e.g., less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least 10 about 35% formamide, e.g., at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, at least about 37° C, or at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of 15 stringency are accomplished by combining these various conditions as needed. In certain embodiments, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In certain embodiments, hybridization will occur at 37° C in 500 mMNaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In certain embodiments, hybridization will occur at 42° C 20 in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art. For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by 25 temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps can be less than about 30 mM NaCl and 3 mM trisodium citrate, e.g., less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, of at least about 42° C, 30 or of at least about 68° C. In certain embodiments, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash steps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In certain embodiments, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those 2024220100   26 Sep 2024 skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Rogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide 5 to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By “substantially identical” or “substantially homologous” is meant a polypeptide or nucleic acid molecule exhibiting at least about 50% homologous or identical to a 0 reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least 15 about 100% homologous or identical to the sequence of the amino acid or nucleic acid used for comparison. Sequence identity can be measured by using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 20   53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, 25 arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e-3 and e-100 indicating a closely related sequence. By “analog” is meant a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule. 30          The term “ligand” as used herein refers to a molecule that binds to a receptor. In certain embodiments, the ligand binds to a receptor on another cell, allowing for cell-to-cell recognition and / or interaction. The term “constitutive expression” or “constitutively expressed” as used herein refers to expression or expressed under all physiological conditions. 2024220100   26 Sep 2024 By “disease” is meant any condition, disease or disorder that damages or interferes with the normal function of a cell, tissue, or organ, e.g., neoplasia, and pathogen infection of cell. By “effective amount” is meant an amount sufficient to have a therapeutic effect. 5 In certain embodiments, an “effective amount” is an amount sufficient to arrest, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion, or migration) of a neoplasia. By “enforcing tolerance” is meant preventing the activity of self-reactive cells or immunoresponsive cells that target transplanted organs or tissues. 10          By “endogenous” is meant a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue. By “exogenous” is meant a nucleic acid molecule or polypeptide that is not endogenously present in a cell, or not present at a level sufficient to achieve the functional effects obtained when over-expressed. The term “exogenous” would therefore 15 encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides. By “exogenous” nucleic acid is meant a nucleic acid not present in a native wild-type cell; for example, an exogenous nucleic acid may vary from an endogenous counterpart by sequence, by position / location, or both. For clarity, an exogenous nucleic 20 acid may have the same or different sequence relative to its native endogenous counterpart; it may be introduced by genetic engineering into the cell itself or a progenitor thereof, and may optionally be linked to alternative control sequences, such as a nonnative promoter or secretory sequence. By a “heterologous nucleic acid molecule or polypeptide” is meant a nucleic acid 25 molecule (e.g., a cDNA, DNA or RNA molecule) or polypeptide that is not normally present in a cell or sample obtained from a cell. This nucleic acid may be from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in a cell or sample. By “immunoresponsive cell” is meant a cell that functions in an immune response 30 or a progenitor, or progeny thereof. By “modulate” is meant positively or negatively alter. Exemplary modulations include a about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100% change. 2024220100   26 Sep 2024 By “increase” is meant to alter positively by at least about 5%. An alteration may be by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more. By “reduce” is meant to alter negatively by at least about 5%. An alteration may 5 be by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even by about 100%. By “isolated cell” is meant a cell that is separated from the molecular and / or cellular components that naturally accompany the cell. The terms “isolated,” “purified,” or “biologically pure” refer to material that is 0 free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause 15 other adverse consequences. That is, a nucleic acid or peptide is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high 20 performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified. 25          The term “antigen-binding domain” as used herein refers to a domain capable of specifically binding a particular antigenic determinant or set of antigenic determinants present on a cell. “Linker”, as used herein, shall mean a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that 30 they are connected to one another. As used herein, a “peptide linker” refers to one or more amino acids used to couple two proteins together (e.g., to couple Vh and Vl domains). In certain embodiments, the linker comprises a sequence set forth in GGGGSGGGGSGGGGS [SEQ ID NO: 31], 2024220100   26 Sep 2024 By “neoplasm” is meant a disease characterized by the pathological proliferation of a cell or tissue and its subsequent migration to or invasion of other tissues or organs. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. 5 Neoplasia can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and 10 vagina, or a tissue or cell type thereof. Neoplasia include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells). In certain embodiments, the neoplasm is a solid tumor. Ilustrative neoplasms for which the presently disclosed subject matter can be used include, but are not limited to leukemias (e.g., acute leukemia, acute lymphocytic 15 leukemia, acute myelocytic leukemia a, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin’s disease, non-Hodgkin’s disease), Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors such as 20 sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, 25 adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm’s tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder 30 carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). 2024220100   26 Sep 2024 By “receptor” is meant a polypeptide, or portion thereof, present on a cell membrane that selectively binds one or more ligand. By “recognize” is meant selectively binds to a target. A T cell that recognizes a tumor can expresses a receptor (e.g., a TCR or CAR) that binds to a tumor antigen. 5          By “reference” or “control” is meant a standard of comparison. For example, the level of scFv-antigen binding by a cell expressing a CAR and an scFv may be compared to the level of scFv-antigen binding in a corresponding cell expressing CAR alone. By “secreted” is meant a polypeptide that is released from a cell via the secretory pathway through the endoplasmic reticulum, Golgi apparatus, and as a vesicle that 0 transiently fuses at the cell plasma membrane, releasing the proteins outside of the cell. By “signal sequence” or “leader sequence” is meant a peptide sequence (e.g., 5, 10, 15, 20, 25 or 30 amino acids) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway. Exemplary leader sequences include, but is not limited to, the IL-2 signal sequence: MYRMQLLSCIALSLALVTNS [SEQ ID NO: 15   12] (human), MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 13] (mouse); the kappa leader sequence: METPAQLLFLLLLWLPDTTG [SEQ ID NO: 14] (human), METDTLLLWVLLLWVPGSTG [SEQ ID NO: 15] (mouse); the CD8 leader sequence: MALPVTALLLPLALLLHAARP [SEQ ID NO: 16] (human); the truncated human CD8 signal peptide: MALPVTALLLPLALLLHA [SEQ ID NO: 28] (human); the albumin 20 signal sequence: MKWVTFISLLFSSAYS [SEQ ID NO: 29] (human); and the prolactin signal sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 30] (human). By “soluble” is meant a polypeptide that is freely diffusible in an aqueous environment (e.g., not membrane bound). By “specifically binds” is meant a polypeptide or fragment thereof that recognizes 25 and binds to a biological molecule of interest (e.g., a polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a presently disclosed polypeptide. The term “tumor antigen” as used herein refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on a tumor cell compared to a normal or non- 30 IS neoplastic cell. In certain embodiments, a tumor antigen includes any polypeptide expressed by a tumor that is capable of activating or inducing an immune response via an antigen recognizing receptor (e.g., CD19, MUC-16) or capable of suppressing an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2). 2024220100   17 Sep 2026 The terms "comprises", "comprising", and are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean "includes", "including" and the like. Unless the context requires otherwise, where the terms “comprise”, “comprises”, “comprised” or “comprising” are used in this specification (including the claims) they are to 5 be interpreted as specifying the presence of the stated features, integers, steps or components, but not precluding the presence of one or more other features, integers, steps or components, or group thereof. As used herein, "treatment" refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for 0 prophylaxis or during the course of clinical pathology. Therapeutic effects of treatment include, without limitation, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastases, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. By preventing progression of a 15 disease or disorder, a treatment can prevent deterioration due to a disorder in an affected or diagnosed subject or a subject suspected of having the disorder, but also a treatment may prevent the onset of the disorder or a symptom of the disorder in a subject at risk for the disorder or suspected of having the disorder. An "individual" or "subject" herein is a vertebrate, such as a human or non-human 20 animal, for example, a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys. The term "immunocompromised" as used herein refers to a subject who has an 25 immunodeficiency. The subject is very vulnerable to opportunistic infections, infections caused by organisms that usually do not cause disease in a person with a healthy immune system, but can affect people with a poorly functioning or suppressed immune system. Other aspects of the presently disclosed subject matter are described in the following disclosure and are within the ambit of the presently disclosed subject matter. 2024220100   17 Sep 2026 2.    HLA-Independent T Cell Receptor (HI-TCR) The present disclosure provides an HI-TCR that binds to an antigen of interest in an HLA-independent manner. In certain non-limiting embodiments, binding of the antigen is capable of activating an immunoresponsive cell comprising the HI-TCR. In certain non- 5 limiting embodiments, the HI-TCR comprises an antigen binding chain. In certain embodiments, the antigen binding chain comprises an extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain is derived from a scFv, Fab, or antibody of murine, human or camelid (e.g., lama) origin. In certain embodiments, the antigen binding chain further comprises a constant domain. 2024220100   26 Sep 2024 2.1. Antigens In certain embodiments, the HI-TCR binds to a tumor antigen. Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigen include, but are not limited to, cancer proteins. The antigen can be 5 expressed as a peptide or as an intact protein or portion thereof. The intact protein or a portion thereof can be native or mutagenized. Non-limiting examples of tumor antigens include carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) 0 infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosineprotein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse 15 transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI,GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY- 20 ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME and ERBB. 25          In certain embodiments, the HI-TCR binds to CD 19. In certain embodiments, the HI-TCR binds to a human CD 19 polypeptide. In certain embodiments, the human CD 19 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. PEEPLWKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQM GGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDR 30 PEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRP ARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWK [SEQ ID NO: 11] . In certain embodiments, the HI-TCR binds to the extracellular domain of a CD 19 protein. 2024220100   26 Sep 2024 In certain embodiments, the HI-TCR binds to a pathogen antigen, e.g., for use in treating and / or preventing a pathogen infection or other infectious disease, for example, in an immunocompromised subject. Non-limiting examples of pathogen includes a virus, bacteria, fungi, parasite and protozoa capable of causing disease. 5          Non-limiting examples of viruses include, Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1 (also referred to as HDTV-III, LAVE or HTLV-IIELAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. 10 equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Fdoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan 15 viruses, bunga viruses, phleboviruses and Naira viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae: Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), 20 herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 =intemally transmitted; class 2 =parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses). 25          Non-limiting examples of bacteria include Pasteur el la. Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria 30 gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium 2024220100   26 Sep 2024 diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani. Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces 5 israelii. In certain embodiments, the pathogen antigen is a viral antigen present in Cytomegalovirus (CMV), a viral antigen present in Epstein Barr Virus (EBV), a viral antigen present in Human Immunodeficiency Virus (HIV), or a viral antigen present in influenza virus. 0   2.2. Extracellular Antigen-Binding Domain In certain embodiments, the antigen binding chain comprises an extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen, e.g., a tumor antigen or a pathogen antigen, e.g., those disclosed in Section 2.1. In certain embodiments, the extracellular antigen-binding 15 domain is capable of dimerizing with another extracellular antigen-binding domain (e.g., forming a fragment variable (Fv)), wherein the dimerized antigen-binding domains (e.g., an Fv) specifically bind to an antigen, e.g., a tumor antigen or a pathogen antigen. In certain embodiments, the extracellular antigen-binding domain comprises a ligand for a cell-surface receptor. In certain embodiments, the extracellular antigen-20 binding domain comprises a receptor for a cell surface ligand. In certain embodiments, the extracellular antigen-binding domain specifically binds to an antigen, e.g., a tumor antigen or a pathogen antigen. In certain embodiments, the antigen binding chain is capable of forming a dimer with another antigen binding chain. In certain embodiments, the HI-TCR comprises a heterodimer comprising two 25 different antigen binding chains. In certain embodiments, the HI-TCR comprises a homodimer comprising two identical antigen binding chains. In certain embodiments, the antigen binding chains dimerize through one or more disulfide-links. In certain embodiments, the antigen binding chain is capable of forming a trimer or oligomer with one or more identical or different antigen binding chains. In certain embodiments, the 30 extracellular antigen-binding domain is capable of dimerizing with another extracellular antigen-binding domain (e.g., forming a fragment variable (Fv)), wherein the dimerized antigen-binding domains (e.g., a Fv) specifically bind to an antigen, e.g., a tumor antigen or a pathogen antigen. 2024220100   26 Sep 2024 In certain non-limiting embodiments, the extracellular antigen-binding domain of the HI-TCR (for example, an Fv or an analog thereof) binds to an antigen with a dissociation constant (Kd) of about 2 x 10'7 M or less. In certain embodiments, the Kais about 2 x 10'7 M or less, about 1 x 10'7 M or less, about 9 x 10'8 M or less, about 1 x 10'8 5 M or less, about 9 x 10'9 M or less, about 5 x 10'9 M or less, about 4 x 10'9 M or less, about 3 x 10'9 or less, about 2 x 10'9 M or less, or about 1 x 10'9 M or less. In certain nonlimiting embodiments, the Kais about 3 x 10'9M or less. In certain non-limiting embodiments, the Kais from about 1 x 10'9 M to about 3 x 10'7M. In certain non-limiting embodiments, the Kais from about 1.5 x 10'9 M to about 3 x 10'7M. In certain non- 10 limiting embodiments, the Kais from about 1.5 x 10'9 M to about 2.7 x 10'7M. Binding of the extracellular antigen-binding domain (for example, a Fv or an analog thereof) can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western Blot assay. Each of these assays generally detect the presence of protein- 15 antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an Fv) specific for the complex of interest. For example, the Fv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is 20 incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography. In certain embodiments, the extracellular antigen-binding domain is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan 25 fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain comprises an antigen binding portion of a TCR. In certain embodiments, the extracellular antigen-binding domain comprises an 30 antigen binding portion of an antibody or a fragment thereof. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (Vh) and / or a light chain variable region (Vl) of an antibody. In certain embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv). In certain embodiments, the extracellular antigen-binding domain comprises a heavy 2024220100   26 Sep 2024 chain-only antibodies (VHH). In certain embodiments, the extracellular antigen-binding domain comprises a Fab, which is optionally crosslinked. In certain embodiments, the extracellular antigen-binding domain comprises a F(ab)2. In certain embodiments, any of the foregoing molecules can be comprised in a fusion protein with a heterologous 5 sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises a heavy chain variable region (Vh) and / or a light chain variable region (Vl) of an antibody, wherein the Vh or the Vl is capable of dimerizing with another extracellular antigenbinding domain comprising a VL or a VH (e.g., forming a fragment variable (Fv)). In 0 certain embodiments, the Fv is a human Fv. In certain embodiments, the Fv is a humanized Fv. In certain embodiments, the Fv is a murine Fv. In certain embodiments, the Fv is identified by screening a Fv phage library with an antigen-Fc fusion protein. Additional extracellular antigen-binding domains targeting an interested antigen can be obtained by sequencing an existing scFv or a Fab region of an existing antibody 15 targeting the same antigen. In certain embodiments, the dimerized extracellular antigen-binding domain of a presently disclosed HI-TCR is a murine Fv. In certain embodiments, the dimerized extracellular antigen-binding domain is an Fv that binds to a human CD 19 polypeptide. In certain embodiments, the extracellular antigen-binding domain is an Fv, which 20 comprises the amino acid sequence of SEQ ID NO: 9 and specifically binds to a human CD19 polypeptide (e.g., a human CD19 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11). In certain embodiments, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 9 is set forth in SEQ ID NO: 10. In certain embodiments, the Fv comprises a heavy chain variable region (Vh) 25 comprising the amino acid sequence set forth in SEQ ID NO: 7. In certain embodiments, the Fv comprises a light chain variable region (Vl) comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the Fv comprises Vh comprising the amino acid sequence set forth in SEQ ID NO: 7 and a Vl comprising the amino acid sequence set forth in SEQ ID NO: 8 . In certain embodiments, the extracellular antigen- 30 binding domain comprises a Vh comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 7. For example, the extracellular antigen-binding domain comprises a Vh comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 2024220100   26 Sep 2024 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 7. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising the amino sequence set forth in SEQ ID NO: 7. In certain embodiments, the 5 extracellular antigen-binding domain comprises a Vl comprising an amino acid sequence that is at least about 80% (e.g, at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 8. For example, the extracellular antigen-binding domain comprises a Vl comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 10   88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises a Vl comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising an amino acid 15 sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 7, and a Vl comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 8. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising the amino acid sequence 20 set forth in SEQ ID NO: 7 and a Vl comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the Fv comprises a heavy chain variable region (Vh) comprising the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the Fv comprises a light chain variable region (Vl) comprising the amino 25 acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the Fv comprises Vh comprising the amino acid sequence set forth in SEQ ID NO: 44 and a Vl comprising the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 30   95%) homologous or identical to SEQ ID NO: 44. For example, the extracellular antigen binding domain comprises a Vh comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to SEQ 2024220100   26 Sep 2024 ID NO: 44. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising the amino sequence set forth in SEQ ID NO: 44. In certain embodiments, the extracellular antigen-binding domain comprises a Vl comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 5   90%, or at least about 95%) homologous to SEQ ID NO: 45. For example, the extracellular antigen-binding domain comprises a Vl comprising an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% 0 homologous or identical to SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain comprises a Vl comprising the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous to SEQ ID NO: 15   44, and a Vl comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to SEQ ID NO: 45. In certain embodiments, the extracellular antigen-binding domain comprises a Vh comprising the amino acid sequence set forth in SEQ ID NO: 44 and a Vl comprising the amino acid sequence set forth in SEQ ID NO: 45. 20          In certain embodiments, the extracellular antigen-binding domain comprises a Vh CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, or a conservative modification thereof, a Vh CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, and a Vh CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a conservative modification thereof. In certain 25 embodiments, the extracellular antigen-binding domain comprises a Vh CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a Vh CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a Vh CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the extracellular antigen-binding domain comprises a Vl CDR1 comprising the amino acid 30 sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, a Vl CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or a conservative modification thereof, and a Vl CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain comprises a Vl CDR1 comprising the amino acid 2024220100   26 Sep 2024 sequence set forth in SEQ ID NO: 4, a Vl CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a Vl CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the extracellular antigen-binding domain comprises a Vh CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or 5 a conservative modification thereof, a Vh CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2 or a conservative modification thereof, a Vh CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a conservative modification thereof, a Vl CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4 or a conservative modification thereof, a Vl CDR2 comprising the amino acid sequence set forth in SEQ 10 ID NO: 5 or a conservative modification thereof, and a Vl CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain comprises a Vh CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a Vh CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a Vh CDR3 comprising 15 the amino acid sequence set forth in SEQ ID NO: 3, a Vl CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a Vl CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5 and a Vl CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6. Table 1 anti-human CD19 scFv (SJ25C1) CDRs 1 2 3 Vh a.a. GYAFSS [SEQ ID NO: 1] YPGDGD [SEQ ID NO: 2] KTISSWDF [SEQ ID NO: 3] Vl a.a. KASQNVGTNVA [SEQ ID NO: 4] SATYRN [SEQ ID NO: 5] QQYNRYPYT [SEQ ID NO: 6] Full Vh EVKLQQSGAE LVRPGSSVKI SCKASGYAFS SYWMNWVKQR PGQGLEWIGQ IYPGDGDTNY NGKFKGQATL TADKSSSTAY MQLSGLTSED SAVYFCARKT ISSWDFYFD YWGQGTTVTV SS [SEQ ID NO: 7] EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDT NYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSWDFYFDYWGQGT TVTV [SEQ ID NO:44] Full Vl DIELTQSPKF MSTSVGDRVS VTCKASQNVG TNVAWYQQKP GQSPKPLIYS ATYRNSGVPD RFTGSGSGTD FTLTITNVQS KDLADYFCQQ YNRYPYTSGG GTKLEIKR [SEQ ID NO: 8] DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGV PDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEI [SEQ ID NO:45] scFv MALPVTALLL PLALLLHAEV KLQQSGAELV RPGSSVKISC KASGYAFSSY WMNWVKQRPG QGLEWIGQIY PGDGDTNYNG KFKGQATLTA DKSSSTAYMQ LSGLTSEDSA VYFCARKTIS SWDFYFDYW GQGTTVTVSS GGGGSGGGGS GGGGSDIELT QSPKFMSTSV GDRVSVTCKA SQNVGTNVAW YQQKPGQSPK PLIYSATYRN SGVPDRFTGS GSGTDFTLTI TNVQSKDLAD YFCQQYNRYP YTSGGGTKLE IKR [SEQ ID NO: 9] DNA ATGGCTCTCCCAGTGACTGCCCTACTGCTTCCCCTAGCGCTTCTCCTGCATGCAGAGG TGAAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTC CTGCAAGGCTTCTGGCTATGCATTCAGTAGCTACTGGATGAACTGGGTGAAGCAGAGG CCTGGACAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGGTGATACTAACT ACAATGGAAAGTTCAAGGGTCAAGCCACACTGACTGCAGACAAATCCTCCAGCACAGC CTACATGCAGCTCAGCGGCCTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGA AAGACCATTAGTTCGGTAGTAGATTTCTACTTTGACTACTGGGGCCAAGGGACCACGG TCACCGTCTCCTCAGGTGGAGGTGGATCAGGTGGAGGTGGATCTGGTGGAGGTGGATC TGACATTGAGCTCACCCAGTCTCCAAAATTCATGTCCACATCAGTAGGAGACAGGGTC AGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGA AACCAGGACAATCTCCTAAACCACTGATTTACTCGGCAACCTACCGGAACAGTGGAGT CCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCACTAAC GTGCAGTCTAAAGACTTGGCAGACTATTTCTGTCAACAATATAACAGGTATCCGTACA CGTCCGGAGGGGGGACCAAGCTGGAGATCAAACGG [SEQ ID NO: 10] 2024220100   26 Sep 2024 As used herein, the term “a conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the presently disclosed HI-TCR comprising the amino acid sequence. 5 Conservative modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced into the Fv of the presently disclosed HI-TCR by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to their physicochemical properties such as charge and polarity. Conservative amino acid 10 substitutions are ones in which the amino acid residue is replaced with an amino acid within the same group. For example, amino acids can be classified by charge: positively-charged amino acids include lysine, arginine, histidine, negatively-charged amino acids include aspartic acid, glutamic acid, neutral charge amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, 15 proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, 20 tryptophan, and valine. Thus, one or more amino acid residues within a CDR region can be replaced with other amino acid residues from the same group and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (1) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues 25 within a specified sequence or a CDR region are altered. 2024220100   26 Sep 2024 The Vh and / or Vl amino acid sequences having at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, 5 or about 99%) homology to a specific sequence (e.g., SEQ ID NOs: 7, and 8) may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the specified sequence(s), but retain the ability to bind to a target antigen (e.g., CD 19). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in a specific sequence (e.g., SEQ ID NOs: 7, and 8). In certain embodiments, 10 substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., in the FRs) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises Vh and / or Vl sequence selected from the group consisting of SEQ ID NOs: 7, and 8, including post-translational modifications of that sequence (SEQ ID NOs: 7 and 8). 15          As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be 20 introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) 25 which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at 30 www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the amino acids sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be 2024220100   26 Sep 2024 performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the specified sequences (e.g., heavy and light chain variable region sequences of scFv m903, 5   m904, m905, m906, and m900) disclosed herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. 0          2.3. Constant Domain In certain embodiments, the antigen binding chain further comprises a constant domain. In certain embodiments, the constant domain comprises a hinge / spacer region and a transmembrane domain. In certain embodiments, the constant domain is capable of forming a homodimer or a heterodimer with another constant domain. In certain 15 embodiments, the constant domain dimerizes through one or more disulfide-links. In certain embodiments, the antigen binding chain is capable of forming a trimer or oligomer with one or more identical or different constant domains. In certain non-limiting embodiments, the constant domain comprises a T cell receptor constant region, e.g., T cell receptor alpha constant region (TRAC), T cell 20 receptor beta constant region (TRBC, e.g., TRBC1 or TRBC2), T cell receptor gamma constant region (TRGC, e.g., TRGC1 or TRGC2), T cell receptor delta constant region (TRDC) or any variants or functional fragments thereof. In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRAC peptide. In certain embodiments, the TRAC 25 polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:38, which is provided below), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative 30 amino acid substitutions. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFAC ANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS [SEQ ID NO:38] 2024220100   26 Sep 2024 In certain embodiments, the TRAC polypeptide has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical to the amino acid sequence encoded by a transcript 5 expressed by the gene of NCBI Genbank ID: 28755, NG_001332.3, range 925603 to 930229 (SEQ ID NO:29, which is provided below), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 1 atatccagaa ccctgaccct gccgtgtacc agctgagaga ctctaaatcc agtgacaagt 10   61 ctgtctgcct attcaccgat tttgattctc aaacaaatgt gtcacaaagt aaggattctg 121 atgtgtatat cacagacaaa actgtgctag acatgaggtc tatggacttc aagagcaaca 181 gtgctgtggc ctggagcaac aaatctgact ttgcatgtgc aaacgccttc aacaacagca 241 ttattccaga agacaccttc ttccccagcc caggtaaggg cagctttggt gccttcgcag 301 gctgtttcct tgcttcagga atggccaggt tctgcccaga gctctggtca atgatgtcta 15   361 aaactcctct gattggtggt ctcggcctta tccattgcca ccaaaaccct ctttttacta 421 agaaacagtg agccttgttc tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag 481 agaaggtggc aggagagggc acgtggccca gcctcagtct ctccaactga gttcctgcct 541 gcctgccttt gctcagactg tttgcccctt actgctcttc taggcctcat tctaagcccc 601 ttctccaagt tgcctctcct tatttctccc tgtctgccaa aaaatctttc ccagctcact 20   661 aagtcagtct cacgcagtca ctcattaacc caccaatcac tgattgtgcc ggcacatgaa 721 tgcaccaggt gttgaagtgg aggaattaaa aagtcagatg aggggtgtgc ccagaggaag 781 caccattcta gttgggggag cccatctgtc agctgggaaa agtccaaata acttcagatt 841 ggaatgtgtt ttaactcagg gttgagaaaa cagctacctt caggacaaaa gtcagggaag 901 ggctctctga agaaatgcta cttgaagata ccagccctac caagggcagg gagaggaccc 25   961 tatagaggcc tgggacagga gctcaatgag aaaggagaag agcagcaggc atgagttgaa 1021 tgaaggaggc agggccgggt cacagggcct tctaggccat gagagggtag acagtattct 1081 aaggacgcca gaaagctgtt gatcggcttc aagcagggga gggacaccta atttgctttt 1141 cttttttttt tttttttttt tttttttttt tgagatggag ttttgctctt gttgcccagg 1201 ctggagtgca atggtgcatc ttggctcact gcaacctccg cctcccaggt tcaagtgatt 30   1261 ctcctgcctc agcctcccga gtagctgaga ttacaggcac ccgccaccat gcctggctaa 1321 ttttttgtat ttttagtaga gacagggttt cactatgttg gccaggctgg tctcgaactc 1381 ctgacctcag gtgatccacc cgcttcagcc tcccaaagtg ctgggattac aggcgtgagc 1441 caccacaccc ggcctgcttt tcttaaagat caatctgagt gctgtacgga gagtgggttg 1501 taagccaaga gtagaagcag aaagggagca gttgcagcag agagatgatg gaggcctggg 35   1561 cagggtggtg gcagggaggt aaccaacacc attcaggttt caaaggtaga accatgcagg 1621 gatgagaaag caaagagggg atcaaggaag gcagctggat tttggcctga gcagctgagt 1681 caatgatagt gccgtttact aagaagaaac caaggaaaaa atttggggtg cagggatcaa 1741 aactttttgg aacatatgaa agtacgtgtt tatactcttt atggcccttg tcactatgta 1801 tgcctcgctg cctccattgg actctagaat gaagccaggc aagagcaggg tctatgtgtg 40   1861 atggcacatg tggccagggt catgcaacat gtactttgta caaacagtgt atattgagta 1921 aatagaaatg gtgtccagga gccgaggtat cggtcctgcc agggccaggg gctctcccta 1981 gcaggtgctc atatgctgta agttccctcc agatctctcc acaaggaggc atggaaaggc 2041 tgtagttgtt cacctgccca agaactagga ggtctggggt gggagagtca gcctgctctg 2101 gatgctgaaa gaatgtctgt ttttcctttt agaaagttcc tgtgatgtca agctggtcga 45   2161 gaaaagcttt gaaacaggta agacaggggt ctagcctggg tttgcacagg attgcggaag 2221 tgatgaaccc gcaataaccc tgcctggatg agggagtggg aagaaattag tagatgtggg 2281 aatgaatgat gaggaatgga aacagcggtt caagacctgc ccagagctgg gtggggtctc 2341 tcctgaatcc ctctcaccat ctctgacttt ccattctaag cactttgagg atgagtttct 2401 agcttcaata gaccaaggac tctctcctag gcctctgtat tcctttcaac agctccactg 50   2461 tcaagagagc cagagagagc ttctgggtgg cccagctgtg aaatttctga gtcccttagg 2521 gatagcccta aacgaaccag atcatcctga ggacagccaa gaggttttgc cttctttcaa 2581 gacaagcaac agtactcaca taggctgtgg gcaatggtcc tgtctctcaa gaatcccctg 2641 ccactcctca cacccaccct gggcccatat tcatttccat ttgagttgtt cttattgagt 2701 catccttcct gtggtagcgg aactcactaa ggggcccatc tggacccgag gtattgtgat 2024220100   26 Sep 2024 2761 gataaattct gagcacctac cccatcccca gaagggctca gaaataaaat aagagccaag 2821 tctagtcggt gtttcctgtc ttgaaacaca atactgttgg ccctggaaga atgcacagaa 2881 tctgtttgta aggggatatg cacagaagct gcaagggaca ggaggtgcag gagctgcagg 2941 cctcccccac ccagcctgct ctgccttggg gaaaaccgtg ggtgtgtcct gcaggccatg 5   3001 caggcctggg acatgcaagc ccataaccgc tgtggcctct tggttttaca gatacgaacc 3061 taaactttca aaacctgtca gtgattgggt tccgaatcct cctcctgaaa gtggccgggt 3121 ttaatctgct catgacgctg cggctgtggt ccagctgagg tgaggggcct tgaagctggg 3181 agtggggttt agggacgcgg gtctctgggt gcatcctaag ctctgagagc aaacctccct 3241 gcagggtctt gcttttaagt ccaaagcctg agcccaccaa actctcctac ttcttcctgt 0   3301 tacaaattcc tcttgtgcaa taataatggc ctgaaacgct gtaaaatatc ctcatttcag 3361 ccgcctcagt tgcacttctc ccctatgagg taggaagaac agttgtttag aaacgaagaa 3421 actgaggccc cacagctaat gagtggagga agagagacac ttgtgtacac cacatgcctt 3481 gtgttgtact tctctcaccg tgtaacctcc tcatgtcctc tctccccagt acggctctct 3541 tagctcagta gaaagaagac attacactca tattacaccc caatcctggc tagagtctcc 5   3601 gcaccctcct cccccagggt ccccagtcgt cttgctgaca actgcatcct gttccatcac 3661 catcaaaaaa aaactccagg ctgggtgcgg gggctcacac ctgtaatccc agcactttgg 3721 gaggcagagg caggaggagc acaggagctg gagaccagcc tgggcaacac agggagaccc 3781 cgcctctaca aaaagtgaaa aaattaacca ggtgtggtgc tgcacacctg tagtcccagc 3841 tacttaagag gctgagatgg gaggatcgct tgagccctgg aatgttgagg ctacaatgag 0   3901 ctgtgattgc gtcactgcac tccagcctgg aagacaaagc aagatcctgt ctcaaataat 3961 aaaaaaaata agaactccag ggtacatttg ctcctagaac tctaccacat agccccaaac 4021 agagccatca ccatcacatc cctaacagtc ctgggtcttc ctcagtgtcc agcctgactt 4081 ctgttcttcc tcattccaga tctgcaagat tgtaagacag cctgtgctcc ctcgctcctt 4141 cctctgcatt gcccctcttc tccctctcca aacagaggga actctcctac ccccaaggag 25   4201 gtgaaagctg ctaccacctc tgtgcccccc cggcaatgcc accaactgga tcctacccga 4261 atttatgatt aagattgctg aagagctgcc aaacactgct gccaccccct ctgttccctt 4321 attgctgctt gtcactgcct gacattcacg gcagaggcaa ggctgctgca gcctcccctg 4381 gctgtgcaca ttccctcctg ctccccagag actgcctccg ccatcccaca gatgatggat 4441 cttcagtggg ttctcttggg ctctaggtcc tgcagaatgt tgtgaggggt ttattttttt 30   4501 ttaatagtgt tcataaagaa atacatagta ttcttcttct caagacgtgg ggggaaatta 4561 tctcattatc gaggccctgc tatgctgtgt atctgggcgt gttgtatgtc ctgctgccga 4621 tgccttc [SEQ ID NO:29] In accordance with the presently disclosed subject matter, an “TRAC nucleic acid molecule” refers to a polynucleotide encoding an TRAC polypeptide. 35          In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRBC peptide. In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRBC2 peptide. In certain embodiments, the TRBC2 polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least 40 about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:39, which is provided below, or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSR 45 YCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSA TILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO:39] In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRBC1 peptide. In certain embodiments, the TRBC1 polypeptide comprises an amino acid sequence that is at least about 80%, at least about 2024220100   26 Sep 2024 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:40, which is provided below, or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative 5 amino acid substitutions. DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDF [SEQ ID NO:40] 10 In certain embodiments, the TRBC polypeptide has an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% homologous or identical to the amino acid sequence encoded by a transcript expressed by a gene of NCBI Genbank ID: 28639, NG_001333.2, range 645749 to 15   647196 (TRBC1, SEQ ID NO: 30), NCBI Genbank ID: 28638, NG 001333.2 range 655095 to 656583 (TRBC2, SEQ ID NO:31) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 1 aggacctgaa caaggtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 61 tctcccacac ccaaaaggcc acactggtgt gcctggccac aggcttcttc cccgaccacg 20   121 tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 181 agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 241 gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 301 acgggctctc ggagaatgac gagtggaccc aggatagggc caaacccgtc acccagatcg 361 tcagcgccga ggcctggggt agagcaggtg agtggggcct ggggagatgc ctggaggaga 25   421 ttaggtgaga ccagctacca gggaaaatgg aaagatccag gtagcagaca agactagatc 481 caaaaagaaa ggaaccagcg cacaccatga aggagaattg ggcacctgtg gttcattctt 541 ctcccagatt ctcagcccaa cagagccaag cagctgggtc ccctttctat gtggcctgtg 601 taactctcat ctgggtggtg ccccccatcc ccctcagtgc tgccacatgc catggattgc 661 aaggacaatg tggctgacat ctgcatggca gaagaaagga ggtgctgggc tgtcagagga 30   721 agctggtctg ggcctgggag tctgtgccaa ctgcaaatct gactttactt ttaattgcct 781 atgaaaataa ggtctctcat ttattttcct ctccctgctt tctttcagac tgtggcttta 841 cctcgggtaa gtaagccctt ccttttcctc tccctctctc atggttcttg acctagaacc 901 aaggcatgaa gaactcacag acactggagg gtggagggtg ggagagacca gagctacctg 961 tgcacaggta cccacctgtc cttcctccgt gccaacagtg tcctaccagc aaggggtcct 35   1021 gtctgccacc atcctctatg agatcctgct agggaaggcc accctgtatg ctgtgctggt 1081 cagcgccctt gtgttgatgg ccatggtaag caggagggca ggatggggcc agcaggctgg 1141 aggtgacaca ctgacaccaa gcacccagaa gtatagagtc cctgccagga ttggagctgg 1201 gcagtaggga gggaagagat ttcattcagg tgcctcagaa gataacttgc acctctgtag 1261 gatcacagtg gaagggtcat gctgggaagg agaagctgga gtcaccagaa aacccaatgg 40   1321 atgttgtgat gagccttact atttgtgtgg tcaatgggcc ctactacttt ctctcaatcc 1381 tcacaactcc tggctcttaa taacccccaa aactttctct tctgcaggtc aagagaaagg 1441 atttctga [SEQ ID NO:30] 1 aggacctgaa aaacgtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 45   61 tctcccacac ccaaaaggcc acactggtat gcctggccac aggcttctac cccgaccacg 121 tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 181 agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 241 gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 2024220100   26 Sep 2024 301 acgggctctc ggagaatgac gagtggaccc aggatagggc caaacccgtc acccagatcg 361 tcagcgccga ggcctggggt agagcaggtg agtggggcct ggggagatgc ctggaggaga 421 ttaggtgaga ccagctacca gggaaaatgg aaagatccag gtagcggaca agactagatc 481 cagaagaaag ccagagtgga caaggtggga tgatcaaggt tcacagggtc agcaaagcac 5   541 ggtgtgcact tcccccacca agaagcatag aggctgaatg gagcacctca agctcattct 601 tccttcagat cctgacacct tagagctaag ctttcaagtc tccctgagga ccagccatac 661 agctcagcat ctgagtggtg tgcatcccat tctcttctgg ggtcctggtt tcctaagatc 721 atagtgacca cttcgctggc actggagcag catgagggag acagaaccag ggctatcaaa 781 ggaggctgac tttgtactat ctgatatgca tgtgtttgtg gcctgtgagt ctgtgatgta 0   841 aggctcaatg tccttacaaa gcagcattct ctcatccatt tttcttcccc tgttttcttt 901 cagactgtgg cttcacctcc ggtaagtgag tctctccttt ttctctctat ctttcgccgt 961 ctctgctctc gaaccagggc atggagaatc cacggacaca ggggcgtgag ggaggccaga 1021 gccacctgtg cacaggtgcc tacatgctct gttcttgtca acagagtctt accagcaagg 1081 ggtcctgtct gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt 5   1141 gctggtcagt gccctcgtgc tgatggccat ggtaaggagg agggtgggat agggcagatg 1201 atgggggcag gggatggaac atcacacatg ggcataaagg aatctcagag ccagagcaca 1261 gcctaatata tcctatcacc tcaatgaaac cataatgaag ccagactggg gagaaaatgc 1321 agggaatatc acagaatgca tcatgggagg atggagacaa ccagcgagcc ctactcaaat 1381 taggcctcag agcccgcctc ccctgcccta ctcctgctgt gccatagccc ctgaaaccct 0   1441 gaaaatgttc tctcttccac aggtcaagag aaaggattcc agaggctag [SEQ ID NO:31] In accordance with the presently disclosed subject matter, an “TRBC nucleic acid molecule” refers to a polynucleotide encoding an TRBC polypeptide. 25          In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRGC peptide. In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRGC1 peptide. In certain embodiments, the TRGC1 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% 30 or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42, which is provided below. DKQLDADVSP KPTIFLPSIA ETKLQKAGTY LCLLEKFFPD VIKIHWQEKK SNTILGSQEG NTMKTNDTYM KFSWLTVPEK SLDKEHRCIV RHENNKNGVD QEIIFPPIKT DVITMDPKDN CSKDANDTLL LQLTNTSAYY MYLLLLLKSV 35         VYFAIITCCL LRRTAFCCNG EKS [SEQ ID NO:42] In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRGC2 peptide. In certain embodiments, the TRGC2 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to 40 the amino acid sequence set forth in SEQ ID NO: 43, which is provided below. DKQLDADVSP KPTIFLPSIA SNTILGSQEG NTMKTNDTYM QEIIFPPIKT DVTTVDPKYN 45        NTSAYYTYLL LLLKSWYFA ETKLQKAGTY LCLLEKFFPD IIKIHWQEKK KFSWLTVPEE SLDKEHRCIV RHENNKNGID YSKDANDVIT MDPKDNWSKD ANDTLLLQLT IITCCLLRRT AFCCNGEKS [SEQ ID NO:43] 2024220100   26 Sep 2024 In certain embodiments, the TRGC polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence encoded by a transcript expressed by a gene of NCBI Genbank ID: 6966, NG_001336.2, range 108270 to 113860 (TRGC1, SEQ ID NO: 32), NCBI Genbank ID: 6967, NG_001336.2, range 124376 to 133924 (TRGC2, SEQ ID NO: 33) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 1 ataaacaact tgatgcagat gtttccccca agcccactat ttttcttcct tcaattgctg 61 aaacaaagct ccagaaggct ggaacatacc tttgtcttct tgagaaattt ttccctgatg 121 ttattaagat acattggcaa gaaaagaaga gcaacacgat tctgggatcc caggagggga 181 acaccatgaa gactaacgac acatacatga aatttagctg gttaacggtg ccagaaaagt 241 cactggacaa agaacacaga tgtatcgtca gacatgagaa taataaaaac ggagttgatc 301 aagaaattat ctttcctcca ataaagacag gtatgtgttt acgcatatca tctgtcagaa 361 cacttctttg aaagtgaatg ctgcattttt tcctttcagt attaatgaaa aacaaacata 421 aatctttctt aaatattgtt acatttaatg gtagcataaa tgccctgcta cttttctata 481 gaattaaaat ggtataggtt ttggagaaaa caaaattgaa aaagttactg aaggtttgtc 541 agcctcagct ccattatcca aaataagaaa gtcacgtgct ggtttttagg gttgttagat 601 ggattaaaga aacaacatac acagaagcat ctagcaacgt gacacgtggt aaacgctcaa 661 aaagtgttct cccttctttt gatgacttta cttgatcagg aaataacata tatatgtctt 721 tcaggaatgt tctgcccaag caggagagtc actcacctca atcttgctac ccacaaagtt 781 taacctaaaa acaacgggtt cattgttgac aaaatgatgt ttatctgttg ttgacagaat 841 gatgtttatc taaaaacagt tccaattttc tatttccttt gctgagacac aaaggggagg 901 caaatgtgca aagcttgagg gtagtcttac cactgtgctt aagtgttctg atttttctag 961 tgatcagggc aaaataaaaa gtatagtaag ttccaaggca gtgaatatta tacaggagag 1021 aagttacagt tttataatgt gttttccttt acactaaatt ctaaaagtaa aaagtctttt 1081 tttttttttg acagagtttc actcttgttg cccaagcagg tgtgctatgg tatgatctca 1141 gctcactgca acctccacct cccgggttca agtgattctc ttacttcagc ctcccgacag 1201 gctgggattg caggcgcctg ccaccacacc tggctaattt ttgtgttttt agtagagatg 1261 gggtttcacc atgttggcca ggctggtctc aaattcctga cctcaagtga tccatccacc 1321 tcggcctcca agtgctggga ttatgggcgt cagccactgt gcccagccta aaagtaaaat 1381 gtctttcatg agcttcccaa ggcagctacg ttaaggagga cacttctctt aatgtcattc 1441 tacagtagat ttctaatgct ctttcttgga agtttgtttt tctgagaaaa gctaaaaata 1501 taacatggaa gtgatcatat tatataatca atgaagtgct tttcaaggag ataaaactaa 1561 tctggtccac acttgcaacc aaccttgatt gagagagaga gagaactcag gatacacttg 1621 aagattttat tatggggaac agttacttta ttctttttac ctcaatcaat gcatggaaat 1681 aagtgatagt cattttcatt tatcttttaa taaatgaagt caccatgagg aaaataaaaa 1741 gacattgaaa acccattaaa gtcagccctt aaagatattt ggacatgcag acttgataac 1801 taacgtttgc attcttgaga cttacccaaa acccatacct caagtccaag tttttagaat 1861 tcatgaaata aagatctcag tgagtgcata aaattgcgca ccagaatcat atccgtatag 1921 acaagaacac atctactaga aaaataataa accaacacac caatgcaact gtgttttctt 1981 ctgttttaaa gtatgttgtc tttgtatgca tgtttgcttc ttcctttttt tttttaacat 2041 cacagataaa ttcaactctc acctcaggtt ttattgagag aactgtcaat gtgacttggc 2101 ctctgtcttt ctagtcccag aaagaattgc actgaaatct gagctcctgt aataaaaaca 2161 accatttgct gagagtaatt aacatactga aagagatttt cttagagtac acaatggtga 2221 cattatattg cctctttata aataactttc tatctatttc tgtggattat tcctacaaag 2281 tacttttcat atgtccaatt tcttttcttc ccctacaact actgtctgaa tactggctct 2341 gctatttgct gatatgattc tcggcaagtt gcctgcactt tttaaacttt atttcctcat 2401 tcagaacatg gggccataca taatacaact cacttcagtg ttattgggga attaaacaaa 2461 aaatgcatgg gaagcattta acatagtgcc tgacacaata atgagtactc agtagatgtt 2521 agcttttatt aatattgttg ttgttatgtc cagaaacact atacctccag aaaatcatgg 2581 gtacttgctg gggacattgg ggatatgcat gatttggaaa agaatgactg ctttttttgc 2641 ttagatgaga aatttttcta agccagactc cttcaaatat gtaagattct gttgtggatt 2701 caaggactga aagaattctt ggccgagtgt ggtggcttat ccctgtaatc ccagcatttt 2761 gtgaggacaa ggcaggaaga ttgcttgagt ccaggagttt gaaaccagcc tgcgcaacat 2821 ggcgaaaccc tgtctctaca aaaaatacaa acattagctc ggagtgagtg ctgacatgtg 2024220100   26 Sep 2024 2881 2941 3001 3061 5   3121 3181 3241 3301 3361 0   3421 3481 3541 3601 3661 5   3721 3781 3841 3901 3961 0   4021 4081 4141 4201 4261 25   4321 4381 4441 4501 4561 30   4621 4681 4741 4801 4861 35   4921 4981 5041 5101 5161 40   5221 5281 5341 5401 5461 45   5521 5581 cctgtactcc gaggcttcag agaccctgcc tgtcatgtca cattgaacaa atacagaagg aatgtattct ctacctaatt aactgtcctc cacacgaaca aaagacaatt cattttgttt gctgcagaat acaaacctaa atagtgatat ctttttcttt gtgcggtggc tgcctcagct tgtattttta ctcgtgatct ccggcctcct tcgagttgtt tatcaagtgc tcttaaaact aataattata atcaaacaaa atagcaagaa gggtgcatgg tgacctacat ttcacagtta ttagcagaca tgcgactaga gctctgactt cagggagtca tggaattctc actcttgtag tgacatcaca catagccata atcagcaaat tagttgggaa ctgacttgtc ggtggtggaa cattaatgta tgcagctcac tctattttgc agaaatcata cagctactca tgagccgtga tcaaaaaaca gcagtgtgtg gtatttatca aaaagtaaat tcaccaactt acacagaatt ctgcctctct attgactagt gttcaaaaga tgaacttctc ggacctagtg ctactgcttt gcttttaatt tctttttttt agtatctcag tctcgagtag gtagagttgg gccccctcgc gtcctgtctt ttgttttagt tttttacaca cctataaaat aaaatcccta gagtttatgt attaccaagc gctttgcaga tttgtgagca ttgaagagat cagaatgagt agcatgggct aggctgccac cggacacatc actttatgaa ctcattgagt tatctcttgc attagaggca ctggattaac tgttctaagt tgactcttgg tatcacttca tcttattctg aaacacctct catcatcacc a gaaggctgag tgacaccgta aacaaacaaa ttgaaggctg aaaacatact gctcttggat caacatcttg agaccaattg gtcgacaggt acttgctctc tgcaaatggt tggcttgaaa atattttaaa ggggtatgag atttcaaaaa ttttttttgg ctcaccacaa ctgggactac ggtttcacca cctcccgaac ttgtttaatg ctttggtctt gaagggcttg ggtacatatt tacatgtcac acttagtaag caggaacctg ctggactttc acagttttct aaaggcatga acttgtttct ctagccctgc accaggcaag acttcttgag gacaaaacat aggcactagt aaaacctcaa tttaggaaag cactttcttt gaatttagca tggtgctggt acgtaaatca taaaaagaac gcatattaca tgctgtctgc atgggaggat ctatactcca caaacaaaac taaagcagcc ttgtggcagt actacactcc atgtgcaaaa tttctggaat tttataaata attcttctag aagcttttgt aatcagggaa ttagtccctc atgactgtaa aaagatttta acagagtctt cctccgcctc aggcgcgtgc tgttggccag tgttgggatt actgggaaaa tgctagtagc ggctgtgttc tttctcccaa acagctagat ttttctgttc aaacaaaacg actccagctc catcagccaa atgtgagatg ggctttttct tcaactttcc gagggcagta atccttttcc ataaatggaa ggtccacccc ataatattaa acaggtgagt gattttccac gaggttgtta agcagtagat tcagaaataa atgtgcaatt tgtacctcct ttagaagaac ctcatgagcc ctccagcctg aaaattaatc atttgttcag cactatgcta agttgtgata acataataca tgtgggctca ttcatttaat atgtcatcac gtttttccct tggattttgc attttctagg attagagagg ttcattcatg gctctgtcac ccggcttcaa caccatgccc gatggcctcg acaggcgtga acatgatacc taataacacg tgcattttct tttacagtcc ctgggatttc tttttctaca gacatcatgt ttttaatgat caaagaataa tctggcatag ctctacatat tctatttcca ccacctcact acaccaagga attttctcag cactgtcttt atgcaatcac gtgccacaac aatgcaacct atcaacttga gtttactttt gtatttgtga tctcttagat cctgctcctc ggctttctgc tggggagttt ggtgacagtg tttttgctga tttatttttc ggagctatga aaaaagaaaa tgaattagat tatttttaat tacacacaca aatggatccc tcctcctgat taggttggat agttgtatta gtacagtggt tgtctttttt ccaggctgga gtgattctcc ggctaatttt aatttgtgac gtcactgtgc atgttgcttc aactagtgtt tgtttaaccc ctttaaagca aaatcaggcc atagagtcag ggggctgggt taggtgtaag ttacaccaga ggcatctcat gcacaaagaa ataccaaggg tgaccaaggg ctgatgtttc gtagagactc acttattcct ccaataatag tacctaacac tactttttaa aagctgaatt aggttttggt acccctctcg acactactgc aagagtgtgg tgcaatggag [SEQ ID NO:32] 1 ataaacaact tgatgcagat gtttccccca agcccactat ttttcttcct tcgattgctg 61 aaacaaaact ccagaaggct ggaacatacc tttgtcttct tgagaaattt ttcccagata 50   121 ttattaagat acattggcaa gaaaagaaga gcaacacgat tctgggatcc caggagggga 181 acaccatgaa gactaacgac acatacatga aatttagctg gttaacggtg ccagaagagt 241 cactggacaa agaacacaga tgtatcgtca gacatgagaa taataaaaac ggaattgatc 301 aagaaattat ctttcctcca ataaagacag gtatgtgttt acacatatca tctgtcagaa 361 cacttctttg aaagtgaatg ctgcattttt tcctttcagt attaatgaaa aacataaatc 55   421 tttcttaaaa attgttacat ttaatggtag cgtaaatgcc ctgctacttt tctatagaat 481 taaaatggta taggttttgg agaaaacaaa attgaaaaag ttgctgaagg tttgtcagcc 541 tcagctccat tatccaaaat aagaaagtca cgtgctggtt tttagggttg ttagatggat 601 taaagaaaca acatacacag aagcatctag caacgtgaca cgtggtaaac gctcaaaaag 661 tgttctccct tcttttgatg actttacttg atcaggaaat aacatatata tgtctttcag 60   721 gaatgttctg cccaagcagg agagtcactc acctcaatct tgctacccac aaagtttaac 781 ctaaaaacaa cgggttcatt gttgacaaaa taatgtttat ctgaagataa ctgtagatca 2024220100   26 Sep 2024 841 tatttatctg tagataatgt ttatctgtgg agtgtggctc tacaaaacat agaatagtct 901 tggtcactgc agttttatag aggccttggg tttttcagag tttcatttta tatatcacca 961 taaagtaaca tttcataatt acaggttggt aaggcttaca tgtacaaaca ttcttccatt 1021 ttccataata aatgcatttc ctgccattgg tgaatgcagc tcaataaaca tttattgtac 5   1081 aattatgaca cgccaggctt agtggaaatg tggatgaaca gacaaggatg agttactgtc 1141 ctaaggatga tgcatgacag tgcagagaat atactctctt cctgatcact cagggtcact 1201 catgattcat gcgcgaggtc ccaaaacagt gcctttgatg cagattctgt acatctctag 1261 acgattggtc caagggctga atgtgctctg gcccagtggt ccagtctgtc actatatgtc 1321 aacatcctga atatgaacat aacagtccaa catctcaaga gtgggcatga aaaggactca 10   1381 ttttgtgctt tttcctgtgg ttaacaagtc ctttttagcc tgggggaaca agcattaaca 1441 aaatgtttga agatctttgc cacgtaccat tccaaatttc tagggtaagt ctttagcttt 1501 tcagatcctg agtttctgca atgatcaaat gtgatttgga cagttgcgtt gactttctcc 1561 tggggctata atggagtgca aaggaaacaa tggcagggaa aatgcttgct ttcaaaatgg 1621 tagcatggat gtgttcattc gtgtagttac tgtattaggt atagcctttc ctgaaactaa 15   1681 ctgaagtggg gttataaaaa cagtcccaat tttctatttc ctttgctgag acacaaagag 1741 gagacaaaag agcaaagctt gagggtagtt ttaccactgt gcttaagtgt tctgattttt 1801 ccagtgatca gggtgaaata aaaagcatag taagttccag ggcagtgaat accatacagg 1861 agacaagtta cagttttata atgtgtttta ctttacacta aattctaaaa gtaaaatgtc 1921 tttttttttt tccgagacag agtttcactc ttgtagccca ggcaggagtg ctatggtgtg 20   1981 atctcggctc acagcaacct ccacctccca gtttcaagcg attcttctgc ctcagcctcc 2041 cgagaagttg aaattacagg tgcctggcac catatctcgc taattattct atttttagta 2101 gagatcgggt tttaccatgt tggccaggct ggtctcgaac tcctgacttc aagtgatcca 2161 cccgcctcag cctcccaaag tgctgggatt acaggtgtga gtcactgtgc cggacctaac 2221 agtaaaatgt ctttcatgtg cttctcaagg caactacatt aaggaggaca catctcttaa 25   2281 tgtcattcta cagtagattt ctaatgctct ttcttggaag tttgtttttc tgagaagagc 2341 taaaaatata ataacatgga agtgatcata ttatataatc aatgaagtgc tttcaaagga 2401 gataaaacta acctggtctg catttgcaac cagccttgat tgagagagag agaactcagg 2461 atacacttag agattttatt atggggaata gttactttat tcattttacc tcaatcaatg 2521 catggaaata agtgacagtc attttcattt atcttttaat aaataaagtc accatgagga 30   2581 aaatgaaaac ccattaaagt cagtccttaa agatatttgg acatgcagac atgataacta 2641 acatttccat tcgtgagact tacccaaaac ctatacctca agtccatttc ttagaataca 2701 tgaaataaag atctcagtga gtgtataaaa ctgcacacca gaatcatatc cgtatagaca 2761 agaatacatc tactagaaaa atataaacca aaacaccaag gtgactctgt ttttttctgt 2821 tttaaaatat gttgtctttg tatgcatgtt tgcttcttcc tttttttttt taaacatcgc 35   2881 agataaattc aactctcacc tcagttgaga gagaactgtc aatgtgactt ggcctctctc 2941 tttctagtcc cagaaagaat tgcactgaaa tgctgagctc ctgtaataaa aatgaccatt 3001 tgctgagagt aattaacata ctgaaagaga ttttcttaga atagtgcaca atggcccaat 3061 ggtgacatta tattgtctct ttataaatta ttttctatct atttctgtgg attatttcta 3121 caaagcactt ttcatatgtc caattccttt tattccccta caagtactga ctgactactg 40   3181 gctctgctgt tcactgatat gactttcggc aagttgcctg cactttttaa acgttatttc 3241 ctcattcaga acatggggcc atacaaaata caactcactt cagtgttatt ggggaattaa 3301 acaaataaat gcatgggaag catttaacat agtgcctgac acaataatga gcactcagta 3361 gatgttagct tttattaata ttgttgttgc tatgtccaga aacactatac ctccagaaaa 3421 tcatgggtac ttgctgggga cgttggggat atgcatgatt ttgaaaggag tgactgctct 45   3481 ttactgctca gatgagaaat ttttctaagc cagactcctt caaacatgta agattctgtt 3541 gtggattcta ggactgaaag aattcttggc cgagtgtggt ggcttatcct ggtaatctca 3601 tcatttggga ggacaaggca ggaagattgc ttgagcccag gagttggaaa caagcctgga 3661 caacatggcg aaaccctgtc tctacaaaaa atacaaacat tagctggtca tgggagtgag 3721 tgcctgtact cccagctact caggaggcta agataggagg atcacctgag cctgggcagt 50   3781 ttgaggtttc agtgagccgt gatgacacca tactatactc cactccagcc tgggtgacag 3841 tgacatcctg cctcaaaaaa acccccaaaa ttattctttt tgctgatttc atgtcagcag 3901 tgtgtgctga aggctgtaaa gtagccactt gttctgttta tttttccatt gaacaagtat 3961 ttatcaaaaa cgtactttgt ggaaggcact gtgctaggaa ctatgcatac agaaggaaaa 4021 ccaaatgttc ttggatacta cactccagtt gtgataaaaa agaaaaaagt attcttcaca 55   4081 aacttcaaca ttttgatgtg caaaaacata atatatgaat tagatctacc taactacaca 4141 gaattagacc aattatttct gggattatgg gctcatattt ttaataactg tcctcctacc 4201 tctctgttga caggttttat aaatattcat ttaattacac acagtcacag acacactcag 4261 acacacacac atacacacac acacacacct tgacaaataa tgggcatgaa caattgactg 4321 gtacttgctc tcattcttct agatgtcacc acagtggatc ccaaatacaa ttattcaaag 60   4381 gatgcaaatg gtaagttttt gtgtttttta tttcctcctg atcattttaa gttttgaact 4441 tctctggctt gaaaaatcag ggaatggatt ttgctaggtt ggatgctgca gaatggacct aatcatattt cttcatgtaa aaaacagact gagtctcgct ccgcctctgg cacatgccac tggccaggct tgggattaca ctcaaggcaa atgctctttc catattgtat caaccaacct ggaatagtta tcatttatct ttaaagttag tgagacttac ctcagtgagt ctagaaaaat ttgtctttgt actctcacct tcccagaaag agtaattaac ctttataaat tccagtttct atgattctcg ccatgtaata aattaaacaa tcagtagatg agaaaatcat tgcttttttc tctgttgtgg atcccagaat gcctgggcaa ggtgtgtgcc tgaagtttga cgacagtgag gtcagcagtg acaagtattt ctaggagcca ataaaaaaga tataaattag catattttta attacacaca tgctctcatt aaatggtaag ggcttgaaaa tgggaggccg gtgaaacccc tagtcccagc tgcagtgagc ccgtctcaaa ttggatgctg gtattaacaa gatatgcttt tttttttttt ggcgcgatct gcttctcaag ttaatagaga tctgccccct tctgtctttt tgttttagtc taaattagtc gagatgactg ttactcatcc ctgttgaaca agttcaagcg catgtccggg ggtctagaat gacgtgagtc ctacgttaag ttggaagttt aatcaatgaa tgattgagag ctttattctt tttaataaat cccttaaaga ccaaaaccca gcataaaatt aataaaccaa atgcatgttt caggttttat aatcgcactg atactgaaag aactttctat tttcttcccc gcaagttgcc ctcatgtacg aaaaatgcat ttagatttta gggtacttgc tgcttagatg attctaggac tttgggagga caagacgaaa tgtattacca ggcttcagtg actctgcgtc tgtgctgaag atcaaaaaca tgaatacaga aaaatgtatt atctacctga ataactgtcc cacacacaca cttctagatg cttttgtgtt atcagggaat aggcgggcgg gtctctacta tacttgggag cgagattgcg aaaaaaaaaa cagaatggac acctaactac cagttatttc tttttttgag cggctcacca tagctgggac tggggtttca cgggctccca gttataatga tttggtcttt cctctttttc taaattgaag atgtgtcttt ggctggattg attctccagc tcatctttgt tcctgacttc actgtgcccg gaggacactt gtttttctga gtgcttttca agagagagaa ttttcctcaa gaagtcacca tatttggaca tacctcaagt gcgcaccaga cacaccaatg gcttcttcct tgagagaact aaatgctgag agattttctt ctatttctgt tacaactacc tgcacttttt tgagtattac ggcaagcatt ttaatattgt ttgggatgtt agaaattttt ggaaagaatt caaggcagga ccctgtctct gctattcggg agccgtgatg aaaaaaaaaa ggtgtaaagt tactttgtgg aggaaaacca cttcacgaac ttacgtagaa tcctgcctct caccttgaca tcatcacaat tttcctttcc gggccgggtg atcacgaggt aaaatacaaa gctgaggcag ccactgcact aaaaaaaaaa ctagtgatat tgcttcgggt aaaaaacaga acggaggagt taacctccgc tacaggtgca ccacgctggc aacttctggg ctggggaaaa gctagtagct taggagttgt ggtacagtga tttcttttct cagtgacgcg ctcagcttct atttttagca gtgatctgcc gcctaacagt ctcttaatgt gaaaagctaa aggagataaa ctcaggatac tcaattcatg tgaggaaaat tgcagacttg ccatgttttt atcatatccg caactgtgtt tttttttttt gtcaatgtga ctcctgtaat agagtacaca ggattattcc gtctgaatac aaactttatt gtaataatgc taacatagtg tgttgttatg ggggatatgc ctaagccaga cttggtcagg agattgcttg acaaaagtac agactgagat acaccatact ccccaaaatt agccacttga tctgtttttg aatgttcttg ttcaacattt tcagaccaat ctgttgacag aataatggac ggatcccaaa tcctgatcat cggtggctca caggagatcg aaattagccg gagaatggcg ccactccagc aagaaaaatc tttaaattag atgagatgac ctttattcat ctcactctat cttactggtt caccaccata caggatggtc attataggcg catgatacca aataacacga attaacaaac tatgctttca tttttttctt atctcacctc caagtagctg gagaccgggt ccctcagccc aaaatgtctt cattctacag aaatataaca actaatctgg acttggagat gaaataagtg aaaaagacat ataactaacg agaattcatg tatagacaag ttcttctgtt taacatcaca cttggcctct aaaaatgacc atggtgacat tacaaagtac tggctctgct tcctcattca tcacttaagt cctgacacaa tccggaaaca atgatttgga ctccttcaaa tgtggtttct agcccaggag ataaattagc gggaggatct atactcgact attgtttttg tcagtttatt ataaataaaa gatactacac tgatatgcaa tatttctgga gttttataaa atgaacaatt gacaattggt tttaagtttt cgcctgtaat agaccatccc ggcttagtgg tgaacccggg ctgggcgaca agggaatgga tccctctttt tgtaaattag ccgtctgtct cacccaggct caagcgattc cctggctaat ttgaattctt tgagccactg tgttgcttct actagtgttt ctaactactg gttatttcaa ttttgagacg actacaacct ggactacagg ttcactatgt tccgaagtgc tcatgcgctt tagatttcta tggaagtgat tccacgtttg tttattatgg atagtcatat tgaaaaccca tttgcattct aaataaagat aacacatcta ttaaaatatg gataaattca gtctttctag atttgctgag tatattgtct ttttcatatg atttgctgat gaacatgggg gttactgggg taatgagcac ctatacctcc aaggtatgac tatgtaagat tatccctgta tttgaaacca ttggcttggt cctgaacctg ccagcctgtg ctgatttcag tttccactga aggcactgtg tccagttgtg aaacatagta attgagggct tattcattta gactagtact caaaagatgc gaacttctct cccagcactt ggctaaaacg cgggcgcctg aggcggagct gagcgagact ttttgctagg tctaggagtt agggtacagt tttttttttt ggagtgcagt tccagcctca ttttgtattt gacctcgtga tgcccggcct tgagttgttt atcaagtgct 2024220100   26 Sep 2024 8161 8221 8281 8341 5   8401 8461 8521 8581 8641 10   8701 8761 8821 8881 8941 15   9001 9061 9121 9181 9241 20   9301 9361 9421 9481 9541 ttttacacag gtagtacaat ccttatacat cgtgcagtta agcaagacat tgcatgagct cctacatttt acagttattg gcagacacag gactagaagc ctgacttagg gggagtcacg gaattctcac tcttgtagct acatcacata tagccataat cagcaaatct gttgggaatg gacttgtctg tggtggaata ttaatatatc cagctcacaa tattttgcca aaatcataa aagggcttgt atattttctc gtcacacagc ctaagtcctc tacaaggcag ttgcagacta gtgagtaaca aagagataag aatgaatact attggctcta ctgccacacc gacacatcac tttatgaaga cattgagtag tctcttgtaa tagaggcatt ggattaacca ttctaagtga actcttggtg tcacttcaac ttattctgta acacctctgc tcatcacctg [SEQ ID NO:33] tctgcatttt ccattttaca taggtctggc tattttttct gaatctgaaa gactttcatt gttttctcat ggcatgaatg tgtttctggc gccctgctca aggcaaggag ttcctgagat caaaacatat gcactagtgg aacctcaaat taggaaagac ctttctttga atttagcaga gtgctggtag gtaaatcatc aaaagaacat atattacacg ctgtctgctt ctagtttaat gtccctttaa atttcaaatc acaatagaaa cgaaagggac ccagctcttt cagccaacta tgagatgtct tttttctctc actttcctct gggcagtacc ccttttccac aaatggaaat tccaccccca aatgttaaat aggtgagtgt ttttccacaa ggttgttaat cagtagatgt agaaataagt gtgcaatttc tacctcctcc agaagaacgg catcttaata agtaaataac aggacatcaa aaatagcaag ataatgtggg taatgattag agaataatta ggcgtagggt tacatatgca atttccaata acctcacttg accaaggact ttctgcagga ctgtctttac gcaatcaccc gccacaacta tgcaacctta caacttgaaa ttacttttag atttgtgaac tcttagatac tgctcctcaa ctttctgctg ctcctataaa tataaaaatc acaaagaatt aattacagat gctgggtggg gtgtaagtga caccagattc atctcattta caaagaatgt ccaaggggct accaagggca gatgtttctg agagactcac ttattccttg aataatagca cctaacacat ctttttaata gctgaattct gttttggtgg ccctctcgca actactgctg gagtgtggtc caatggagag In accordance with the presently disclosed subject matter, an “TRGC nucleic acid molecule” refers to a polynucleotide encoding an TRGC polypeptide. In certain embodiments, the constant domain of a presently disclosed HI-TCR comprises a native or modified TRDC peptide. In certain embodiments, the TRDC 30 polypeptide has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 41, which is provided below. SQPHTKPSVF VMKNGTNVAC LVKEFYPKDI RINLVSSKKI TEFDPAIVIS 35         PSGKYNAVKL GKYEDSNSVT CSVQHDNKTV HSTDFEVKTD STDHVKPKET ENTKQPSKSC HKPKAIVHTE KVNMMSLTVL GLRMLFAKTV AVNFLLTAKL FFL [SEQ ID NO:41] In certain non-limiting embodiments, T cell receptor constant region comprises a hinge / spacer region that links the extracellular antigen-binding domain to the constant 40 domain. The hinge / spacer region can be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition. In certain nonlimiting embodiments, the hinge / spacer region can be the hinge region from IgGl, or the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, a variation of any of the foregoing which is 45 at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical thereto, or a synthetic spacer sequence. In certain non-limiting 2024220100   26 Sep 2024 embodiments, the hinge / spacer region of the CAR can comprise a native or modified hinge region of a CD3^ polypeptide, a CD40 polypeptide, a 4-IBB polypeptide, an OX40 polypeptide, a CD 166 peptide, a CD 166 peptide, a CD8a peptide, a CD8b peptide, an ICOS polypeptide, an ICAM-1 peptide, a CTLA-4 peptide, a synthetic peptide (not based 5 on a protein associated with the immune response), or a combination thereof. 2.4.   Intracellular Signaling Domain In certain non-limiting embodiments, a presently disclosed HI-TCR comprises an antigen binding chain, which does not comprise an intracellular domain. In certain embodiments, the antigen binding chain is capable of associating with a CD3(^ 0 polypeptide. In certain embodiments, the antigen binding chain comprises a constant domain, which is capable of associating with a CD3(^ polypeptide. In certain embodiments, the CD3(^ polypeptide is endogenous. In certain embodiments, the CD3(^ polypeptide is exogenous. In certain embodiments, binding of the antigen binding chain to an antigen is capable of activating the CD3(^ polypeptide associated to the antigen 15 binding chain. In certain embodiments, the exogenous CD3(^ polypeptide is fused to or integrated with a costimulatory molecule disclosed herein. In certain non-limiting embodiments, a presently disclosed HI-TCR comprises an antigen binding chain that comprises an intracellular domain. In certain embodiments, the intracellular domain comprises a CD3(^ polypeptide. In certain embodiments, binding 20 of the antigen binding chain to an antigen is capable of activating the CD3(^ polypeptide of the antigen binding chain. The activated CD3 £ polypeptide can activate and / or stimulate an immunoresponsive cell (e.g., a cell of the lymphoid lineage, e.g., a T cell). CD3(^ comprises three immunoreceptor tyrosine-based activation motifs (ITAMI, ITAM2 and 25 ITAM3), three basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3), and transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T cell) after antigen is bound to the antigen binding chain. The intracellular signaling domain of the CD3^-chain is the primary transmitter of signals from endogenous TCRs. In certain embodiments, the CD3(^ polypeptide comprises or has an amino acid sequence that is at 30 least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous to the sequence having a NCBI Reference No: NP_932170 (SEQ ID NO: 17), NCBI Reference No: NP_000725.1 (SEQ ID NO: 18) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain non-limiting embodiments, the CD3(^ polypeptide 2024220100   26 Sep 2024 comprises or has an amino acid sequence that is a consecutive portion of SEQ ID NO: 17, which is at least 20, or at least 30, or at least 40, or at least 50, and up to 164 amino acids in length. Alternatively or additionally, in non-limiting various embodiments, the CD3(^ polypeptide comprises or has an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 5 to 100, 100 to 150, or 150 to 164 of SEQ ID NO: 17. In certain embodiments, the CD3(^ polypeptide comprises or has an amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 17. SEQ ID NO: 17 is provided below: 1 MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF IYGVILTALF LRVKFSRSAD 10    61 APAYQQGQNQ LYNELNLGRR EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA 121 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR [SEQ ID NO: 17] In certain embodiments, the intracellular signaling domain comprises a human CD3(^ polypeptide. The human CD3(^ polypeptide can comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 15   98%, about 99% or about 100% homologous or identical to SEQ ID NO: 18 or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 18 is provided below: RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP PR [SEQ ID NO: 20    18] . An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 18 is set forth in SEQ ID NO: 19, which is provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG 25 CCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCC ACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 19] 2.4.1. Co-stimulatory region In certain non-limiting embodiments, a presently disclosed HI-TCR comprises an 30 antigen binding chain that comprises an intracellular domain, wherein the intracellular domain comprises a co-stimulatory region. In certain embodiments, the intracellular domain comprises a co-stimulatory region and a CD3(^ polypeptide. In certain embodiments, the intracellular domain comprises a co-stimulatory region and does not comprise a CD3(^ polypeptide. 2024220100   26 Sep 2024 In certain embodiments, the co-stimulatory region comprises at least one costimulatory molecule, which can provide optimal lymphocyte activation. As used herein, “co-stimulatory molecules” refer to cell surface molecules other than antigen receptors or their ligands that are required for an efficient response of lymphocytes to antigen. The at 5 least one co-stimulatory signaling region can include a CD28 polypeptide, a 4-IBB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. The co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on cell surface that upon binding to its receptor produces a co-stimulatory response, i.e., an intracellular response that effects the stimulation 0 provided when an antigen binds to its CAR molecule. Co-stimulatory ligands, include, but are not limited to CD80, CD86, CD70, OX40L, and 4-1BBL. As one example, a 4-1BB ligand (i.e., 4-1BBL) may bind to 4-1BB (also known as “CD137”) for providing an intracellular signal that in combination with a CAR signal induces an effector cell function of the CAR+ T cell. CARs comprising an intracellular signaling domain that 15 comprises a co-stimulatory signaling region comprising 4-1BB, ICOS or DAP-10 are disclosed in U.S. 7,446,190, which is herein incorporated by reference in its entirety. In certain embodiments, the co-stimulatory region of an antigen binding chain of a HI-TCR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide. The CD28 polypeptide can comprise or have an amino acid sequence that is at least about 20   85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the sequence having a NCBI Reference No: Pl0747 or NP 006130 (SEQ ID NO: 20), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In non-limiting certain embodiments, the CD28 polypeptide comprises or has an amino acid sequence 25 that is a consecutive portion of SEQ ID NO: 20 which is at least 20, or at least 30, or at least 40, or at least 50, and up to 220 amino acids in length. Alternatively or additionally, in non-limiting various embodiments, the CD28 polypeptide comprises or has an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, or 200 to 220 of SEQ ID NO: 20. In certain embodiments, the co-stimulatory region 30 comprises a co-stimulatory signaling region that comprises a CD28 polypeptide comprising or having an amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 20. 1 MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC KYSYNLFSRE FRASLHKGLD 61 SAVEVCWYG NYSQQLQVYS KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP 2024220100   26 Sep 2024 121 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVWG GVLACYSLLV TVAFIIFWVR 181 SKRSRLLHSD YMNMTPRRPG PTRKHYQPYA PPRDFAAYRS [SEQ ID NO: 20] In certain embodiments, the co-stimulatory region comprises a human intracellular signaling domain of CD28. The human intracellular signaling domain of CD28 can 5 comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO: 21 or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 21 is provided below: 10 RSKRSRLLHS DYMNMTPRRP GPTRKHYQPY APPRDFAAYR S [SEQ ID NO: 21]. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21 is set forth in SEQ ID NO: 22, which is provided below. AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGC AAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 22] 15          In certain embodiments, the co-stimulatory region comprises a co-stimulatory signaling region that comprises two co-stimulatory molecules, e.g., co-stimulatory signaling regions of CD28 and 4-IBB or co-stimulatory signaling regions of CD28 and OX40. 4-IBB can act as a tumor necrosis factor (TNF) ligand and have stimulatory 20 activity. The 4-IBB polypeptide can comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having a NCBI Reference No: P41273 or NP_001552 (SEQ ID NO: 23) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 25          SEQ ID NO: 23 is provided below: 1   MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR 61  TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC 121 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDWCGP SPADLSPGAS SVTPPAPARE 181 PGHSPQIISF FLALTSTALL FLLFFLTLRF SWKRGRKKL LYIFKQPFMR PVQTTQEEDG 30   241 CSCRFPEEEE GGCEL [SEQ ID NO: 23] In accordance with the presently disclosed subject matter, a “4-1BB nucleic acid molecule” refers to a polynucleotide encoding a 4-IBB polypeptide. In certain embodiments, the co-stimulatory region comprises an intracellular signaling domain of 4-1BB. The intracellular signaling domain of 4-1BB can comprise or 35 have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 2024220100   26 Sep 2024 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to SEQ ID NO: 24 or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 24 is provided below: KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL [SEQ ID NO: 24]. 5         An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 is set forth in SEQ ID NO: 27, which is provided below. AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCA AGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG [SEQ ID NO: 27] An OX40 polypeptide can comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to the sequence having a NCBI Reference No: P43489 or NP 003318 (SEQ ID NO: 25), or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 15          SEQ ID NO: 25 is provided below: 1   MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 61  NTVCRPCGPG FYNDWSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 20   241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI [SEQ ID NO: 25] In accordance with the presently disclosed subject matter, an “OX40 nucleic acid molecule” refers to a polynucleotide encoding an OX40 polypeptide. An ICOS polypeptide can comprise or have an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 25 about 100% homologous or identical to the sequence having a NCBI Reference No: NP 036224 (SEQ ID NO: 26) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 26 is provided below: 1   MKSGLWYFFL FCLRIKVLTG EINGSANYEM FIFHNGGVQI LCKYPDIVQQ FKMQLLKGGQ 30   61  ILCDLTKTKG SGNTVSIKSL KFCHSQLSNN SVSFFLYNLD HSHANYYFCN LSIFDPPPFK 121 VTLTGGYLHI YESQLCCQLK FWLPIGCAAF VWCILGCIL ICWLTKKKYS SSVHDPNGEY 181 MFMRAVNTAK KSRLTDVTL [SEQ ID NO: 26] In accordance with the presently disclosed subject matter, an “ICOS nucleic acid molecule” refers to a polynucleotide encoding an ICOS polypeptide. 35          In certain embodiments, mutation sites and / or junction between domains / motifs / regions of the CAR derived from different proteins are de-immunized. 2024220100   26 Sep 2024 Immunogenicity of junctions between different CAR moieties can be predicted using NetMHC 4.0 Server. For each peptide containing at least 1 aa from next moiety, binding affinity to HLA A, B and C, for all alleles, can be predicted. A score of immunogenicity of each peptide can be assigned for each peptide. Immunogenicity score can be calculated 5 using the formula Immunogenicity score=[(50-binding affinity)*HLA frequency]n. n is the number of prediction for each peptide. 2.5. CD3 Complex In certain embodiments, a presently disclosed HI-TCR is capable of associating with a CD3 complex (also known as “T-cell co-receptor”). In certain embodiments, the 10 HI-TCR and the CD3 complex form an antigen recognizing receptor complex similar to a native TCR / CD3 complex. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the CD3 complex is exogenous. In certain embodiments, the presently disclosed HI-TCR replaces a native and / or an endogenous TCR in the CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3y chain, 15 a CD36 chain, and two CD3s chains. In certain embodiments, the CD3y chain comprises or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to NCBI reference number: NP_000064.1 (SEQ ID NO: 34, which is provided below) or fragments thereof, and / or 20 may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 1 meqgkglavl ilaiillqgt laqsikgnhl vkvydyqedg svlltcdaea knitwfkdgk 61 migfltedkk kwnlgsnakd prgmyqckgs qnkskplqvy yrmcqnciel naatisgflf 121 aeivsifvla vgvyfiagqd gvrqsrasdk qtllpndqly qplkdreddq yshlqgnqlr 25   181 rn [SEQ ID NO:34] In certain embodiments, the CD36 chain comprises or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% homologous or identical to NCBI reference numbers: 30   NP_000723.1 (SEQ ID NO: 35, which is provided below), NP_001035741.1 (SEQ ID NO: 36, which is provided below) or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 1 mehstflsgl vlatllsqvs pfkipieele drvfvncnts itwvegtvgt llsditrldl 61 gkrildprgi yrcngtdiyk dkestvqvhy rmcqscveld patvagiivt dviatlllal 35   121 gvfcfaghet grlsgaadtq allrndqvyq plrdrddaqy shlggnwarn k [SEQ ID NO: 35] 1 mehstflsgl vlatllsqvs pfkipieele drvfvncnts itwvegtvgt llsditrldl 2024220100   26 Sep 2024 61 gkrildprgi yrcngtdiyk dkestvqvhy rtadtqallr ndqvyqplrd rddaqyshlg 121 gnwarnk [SEQ ID NO: 36] In certain embodiments, the CD3s chain comprises or has an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, 5 about 99% or about 100% homologous or identical to NCBI reference number: NP_000724.1 (SEQ ID NO: 37, which is provided below)or fragments thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. 1 mqsgthwrvl glcllsvgvw gqdgneemgg itqtpykvsi sgttviltcp qypgseilwq 0   61 hndkniggde ddknigsded hlslkefsel eqsgyyvcyp rgskpedanf ylylrarvce 121 ncmemdvmsv ativivdici tggllllvyy wsknrkakak pvtrgagagg rqrgqnkerp 181 ppvpnpdyep irkgqrdlys glnqrri [SEQ ID NO: 37] In certain embodiments, the recombinant TCR exhibits a greater antigen 5 sensitivity than a CAR targeting the same antigen. In certain embodiments, the recombinant TCR is capable of inducing an immune response when binding to an antigen that has a low density on the surface of a tumor cell. In certain embodiments, immunoresponsive cells comprising a presently disclosed HI-TCR can be used to treat a subject having tumor cells with a low expression level of a surface antigen, e.g., from a 20 relapse of a disease, wherein the subject received treatment which leads to residual tumor cells. In certain embodiments, the tumor cells have a low density of a target molecule on the surface of the tumor cells. In certain embodiments, a target molecule having a low density on the cell surface has a density of less than about 5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than 25 about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, a target molecule having a low density on the cell surface has a density of less than about 2,000 molecules per cell. In certain embodiments, a target molecule having a low density 30 on the cell surface has a density of less than about 1,500 molecules per cell. In certain embodiments, a target molecule having a low density on the cell surface has a density of less than about 1,000 molecules per cell. In certain embodiments, a target molecule having a low density on the cell surface has a density of between about 4,000 molecules per cell and about 2,000 molecules per cell, between about 2,000 molecules per cell and 35 about 1,000 molecules per cell, between about 1,500 molecules per cell and about 1,000 molecules per cell, between about 2,000 molecules per cell and about 500 molecules per 2024220100   26 Sep 2024 cell, between about 1,000 molecules per cell and about 200 molecules per cell, or between about 1,000 molecules per cell and about 100 molecules per cell. 2.6. HI-TCR 19 In certain embodiments, a presently disclosed HI-TCR comprises two antigen 5 binding chains, e.g., VL-TRAC and VH-TRBC, which are capable of dimerizing, wherein the HI-TCR binds to CD19 (e.g., human CD19). VL-TRAC In certain embodiments, a presently disclosed HI-TCR comprises an antigen binding chain that comprises an extracellular antigen-binding domain of a Vl domain of 10 an antibody and a constant domain of TRAC. In certain embodiments, the antibody binds to CD 19 (e.g., human CD 19). In certain embodiments, the antigen binding chain is designated as “VL-TRAC”. VH-TRBC In certain embodiments, a presently disclosed HI-TCR comprises an antigen 15 binding chain that comprises an extracellular antigen-binding domain of a Vh domain of an antibody and a constant domain of TRBC. In certain embodiments, the antibody binds to CD 19 (e.g., human CD 19). In certain embodiments, the antigen binding chain is designated as “VH-TRBC”. 3. Immunoresponsive Cells 20          The presently disclosed subject matter provides immunoresponsive cells comprising a presently disclosed HI-TCR. In certain embodiments, the HI-TCR is capable of activating the immunoresponsive cell. Upon binding to the antigen, the immunoresponsive cells exhibit cytolytic effects towards cells bearing the antigen. In certain embodiments, the immunoresponsive cells comprising the HI-TCR exhibits 25 comparable or better therapeutic potency compared to cells comprising a chimeric antigen receptor (CAR) targeting the same antigen. In certain embodiments, the immunoresponsive cells comprising the HI-TCR exhibit comparable or better cytolytic effects compared to cells comprising a chimeric antigen receptor (CAR) targeting the same antigen. In certain embodiments, the immunoresponsive cells comprising the HI- 30 TCR secrete anti-tumor cytokines. The cytokines secreted by the immunoresponsive cells include, but are not limited to, TNFa, IFNy and IL2. The immunoresponsive cells of the presently disclosed subject matter can be cells of the lymphoid lineage. The lymphoid lineage, comprising B, T and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, 2024220100   26 Sep 2024 detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immunoresponsive cells of the lymphoid lineage include T cells, Natural Killer T (NKT) cells, and precursors thereof including embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells may be 5 differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and 0 two types of effector memory T cells: e.g., Tem cells and Temra cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and y6 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient’s own T cells may be genetically modified to target specific antigens through the 15 introduction of an HI-TCR. In certain embodiments, the immunoresponsive cell is a T cell. The T cell can be a CD4+ T cell or a CD8+ T cell. In certain embodiments, the T cell is a CD4+ T cell. In certain embodiments, the T cell is a CD8+ T cell. In certain embodiments, the immunoresponsive cell comprises an exogenous or a recombinant (e.g., the cell is transduced with) at least one co-stimulatory ligand. In 20 certain embodiments, the immunoresponsive cell co-expresses the HI-TCR and the at least one exogenous co-stimulatory ligand. The interaction between the HI-TCR and at least one exogenous co-stimulatory ligand provides a non-antigen-specific signal important for full activation of an immunoresponsive cell (e.g, T cell). Co-stimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) 25 superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic 30 segment and a relatively long extracellular region. TNF superfamily members include, but are not limited to, nerve growth factor (NGF), CD40L (CD40L) / CD154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), 2024220100   26 Sep 2024 lymphotoxin-beta (LTP), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), and TNF-related apoptosisinducing ligand (TRAIL), LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, 5 binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins — they possess an immunoglobulin domain (fold). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD275, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, 10 and combinations thereof. In certain embodiments, the immunoresponsive cell comprises or consists of one exogenous or recombinant co-stimulatory ligand. In certain embodiments, the one exogenous or recombinant co-stimulatory ligand is 4-1BBL or CD80. In certain embodiments, the one exogenous or recombinant co-stimulatory ligand is 4-1BBL. In certain embodiments, the immunoresponsive cell comprises or consists of 15 two exogenous or recombinant co-stimulatory ligands. In certain embodiments, the two exogenous or recombinant co-stimulatory ligands are 4-1BBL and CD80. In certain embodiments, the immunoresponsive cell can comprise or be transduced with at least one chimeric co-stimulatory receptor (CCR). As used herein, the term “chimeric co-stimulatory receptor” or “CCR” refers to a chimeric receptor that binds 20 to an antigen, and, upon its binding to the antigen, provides a co-stimulatory signal to a cell (e.g., a T cell) comprising the CCR, but does not alone provide an activation signal to the cell. CCR is described in Krause, et al., J. Exp. Med. (1998);188(4):619-626, and US20020018783, which is incorporated by reference in its entirety. CCRs mimic costimulatory signals, but unlike, CARs, do not provide a T-cell activation signal, e.g., 25 CCRs lack a CD3(^ polypeptide. CCRs provide co-stimulation, e.g., a CD28-like signal, in the absence of the natural co-stimulatory ligand on the antigen-presenting cell. A combinatorial antigen recognition, i.e., use of a CCR in combination with a CAR, can augment T-cell reactivity against the dual-antigen expressing T cells, thereby improving selective tumor targeting. See WO2014 / 055668, which is incorporated by reference in its 30 entirety. Kloss et al., describe a strategy that integrates combinatorial antigen recognition, split signaling, and, critically, balanced strength of T-cell activation and costimulation to generate T cells that eliminate target cells that express a combination of antigens while sparing cells that express each antigen individually (Kloss et al., Nature Biotechnololgy (2013);31(1):71-75, the content of which is incorporated by reference in 2024220100   26 Sep 2024 its entirety). With this approach, T-cell activation requires CAR-mediated recognition of one antigen, whereas co-stimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, the combinatorial antigen recognition approach diminishes the efficiency of T-cell activation to a level where it is ineffective 5 without rescue provided by simultaneous CCR recognition of the second antigen. In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds to a second antigen, a transmembrane domain, and a co-stimulatory signaling region that comprises at least one co-stimulatory molecule. In certain embodiments, the CCR does not alone deliver an activation signal to the cell. Non- 0 limiting examples of co-stimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10 and any combination thereof. In certain embodiments, the co-stimulatory signaling region of the CCR comprises one co-stimulatory signaling molecule. In certain embodiments, the one co-stimulatory signaling molecule is CD28. In certain embodiments, the one co-stimulatory signaling molecule is 4-1BB. In certain 15 embodiments, the co-stimulatory signaling region of the CCR comprises two costimulatory signaling molecules. In certain embodiments, the two co-stimulatory signaling molecules are CD28 and 4-1BB. A second antigen is selected so that expression of both the first antigen and the second antigen is restricted to the targeted cells (e.g., cancerous tissue or cancerous cells). Similar to a CAR, the extracellular 20 antigen-binding domain can be a scFv, a Fab, a F(ab)2, or a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the CCR is co-expressed with a HI-TCR binding to an antigen that is different from the antigen to which the CCR binds, e.g., the HI-TCR binds to a first antigen and the CCR binds to a second antigen. 25          Types of human lymphocytes of the presently disclosed subject matter include, without limitation, peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), in Morgan, R.A., etal. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen- 30 recognizing T cell receptor complex comprising the a and P heterodimer), in Panelli, M.C., et al. 2000 J Immunol 164:495-504; Panelli, M.C., etal. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies), and in Dupont, J., et al. 2005 Cancer Res 2024220100   26 Sep 2024 65:5417-5427; Papanicolaou, G.A., etal. 2003 Blood 102:2498-2505 (disclosing selectively in vitro-Qx^andQd antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells). The immunoresponsive cells (e.g., T cells) can be autologous, non-autologous (e.g., 5 allogeneic), or derived in vitro from engineered progenitor or stem cells. The presently disclosed immunoresponsive cells are capable of modulating the tumor microenvironment. Tumors have a microenvironment that is hostile to the host immune response involving a series of mechanisms by malignant cells to protect themselves from immune recognition and elimination. This “hostile tumor 10 microenvironment” comprises a variety of immune suppressive factors including infiltrating regulatory CD4+ T cells (Tregs), myeloid derived suppressor cells (MDSCs), tumor associated macrophages (TAMs), immune suppressive cytokines including TGF-P, and expression of ligands targeted to immune suppressive receptors expressed by activated T cells (CTLA-4 and PD-1). These mechanisms of immune suppression play a 15 role in the maintenance of tolerance and suppressing inappropriate immune responses, however within the tumor microenvironment these mechanisms prevent an effective antitumor immune response. Collectively these immune suppressive factors can induce either marked anergy or apoptosis of adoptively transferred CAR modified T cells upon encounter with targeted tumor cells. 20          The unpurified source of CTLs may be any known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-CTLs initially. mAbs are particularly useful for identifying markers associated with particular cell lineages 25 and / or stages of differentiation for both positive and negative selections. A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. In certain embodiments, at least about 80%, usually at least about 70% of the total hematopoietic cells will be removed prior to cell 30 isolation. Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, 2024220100   26 Sep 2024 complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g. plate, chip, elutriation or any other convenient technique. Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color 5 channels, low angle and obtuse light scattering detecting channels, impedance channels. The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). In certain embodiments, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, e.g., sterile, isotonic medium. 0   4. Vectors Genetic modification of an immunoresponsive cell (e.g., a T cell or an NKT cell) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (either gamma-retroviral or lentiviral) is employed for the introduction of the DNA construct into 15 the cell. For example, a polynucleotide encoding an HI-TCR can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Non-viral vectors may be used as well. For initial genetic modification of an immunoresponsive cell to include an HI-20 TCR, a retroviral vector is generally employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. The HI-TCR can be constructed with an auxiliary molecule (e.g., a cytokine) in a single, multi ci stronic expression cassette, in multiple expression cassettes of a single vector, or in multiple vectors. Examples of elements that create polycistronic expression cassette include, but is 25 not limited to, various viral and non-viral Internal Ribosome Entry Sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picomavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides , e.g., P2A, T2A, E2A and F2A peptides). Combinations of 30 retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virusproducing cell lines are known, including, but not limited to, PA12 (Miller, etal. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, etal. (1986)Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non- 2024220100   26 Sep 2024 amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art. Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method ofBregni, etal. (1992) Blood 80:1418-1422, or 5 culturing with viral supernatant alone or concentrated vector stocks with or without appropriate growth factors and polycations, e.g., by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, etal. (1992) J. Clin. Invest. 89:1817. Other transducing viral vectors can be used to modify an immunoresponsive cell. In certain embodiments, the chosen vector exhibits high efficiency of infection and stable 10 integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adena-associated viral vectors, vaccinia 15 virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; 20 Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S- 83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). 25          Non-viral approaches can also be employed for genetic modification of an immunoresponsive cell. For example, a nucleic acid molecule can be introduced into an immunoresponsive cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., 30 Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and 2024220100   26 Sep 2024 protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell 5 (or its descendants) are injected into a targeted tissue or are injected systemically. Recombinant receptors can also be derived or obtained using transposases or targeted nucleases (e.g. Zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression may be obtained by RNA electroporation. In certain embodiments, recombinant receptors can be introduced by a transposon-based vector. In certain 0 embodiments, the transposon-based vector comprises a transposon (a.k.a. a transposable element). In certain embodiments, the transposon can be recognized by a transposase. In certain embodiments, the transposase is a Sleeping Beauty transposase. Clustered regularly-interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When utilized for genome editing, 15 the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9), trans-activating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair 20 template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence). CRISPR / Cas9 often employs a plasmid to transfect the target cells. The crRNA needs to be designed for each application as this is the sequence that Cas9 uses to identify and directly bind to the target DNA in a cell. The repair template carrying CAR expression cassette need also be designed for each application, as it must overlap with the 25 sequences on either side of the cut and code for the insertion sequence. Multiple crRNA's and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells. A zinc-finger nuclease (ZFN) is an artificial restriction enzyme, which is 30 generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific DNA sequences which allows a zinc-finger nuclease to target desired sequences within genomes. The DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of basepairs. The most common method to 2024220100   26 Sep 2024 generate new zinc-finger domain is to combine smaller zinc-finger "modules" of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type Ils restriction endonuclease Fokl. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying 5 CAR expression cassette, ZFNs can be used to insert the CAR expression cassette into genome. When the targeted sequence is cleaved by ZFNs, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, whereby the homologous DNA template is integrated into the genome. 10          Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid 15 repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus 20 (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g. the elongation factor la enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those 25 that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above. The resulting cells can be grown under conditions similar to those for unmodified 30 cells, whereby the modified cells can be expanded and used for a variety of purposes. 6. Genome Editing Methods In certain non-limiting embodiments, an HI-TCR, a costimulatory ligand, a CCR or any other molecule / transgene disclosed herein is expressed by an immunoresponsive cell through a modified genomic locus. In certain embodiments, an expression cassette of 2024220100   26 Sep 2024 the transgene is integrated into a targeted genomic locus of an immunoresponsive cell through targeted genome editing methods. In certain embodiments, the targeted genomic locus can be CD33, CD3e, CD247, B2M, TRAC, TRBC1, TRBC2, TRGC1 and / or TRGC2 loci. 5          6.1. Engineering T Cell Receptor Locus In certain embodiments, an HI-TCR is expressed by an immunoresponsive cell through a modified endogenous T cell receptor locus. In certain embodiments, an HI-TCR expression cassette is integrated at an endogenous T cell receptor locus. In certain embodiments, the HI-TCR expression cassette is integrated within the T cell receptor 0 alpha locus (TRA, GenBank ID: 6955). In certain embodiments, the HI-TCR expression cassette is integrated within the T cell receptor beta locus (TRB, GenBank ID: 6957). In certain embodiments, the HI-TCR expression cassette is integrated within the T cell receptor gamma locus (TRG, GenBank ID: 6965). In certain embodiments, the HI-TCR expression cassette comprises an 15 extracellular antigen-binding domain that is integrated in the first exon of a TCR constant domain locus, so that the extracellular antigen-binding domain and the TCR constant domain are comprised in one antigen binding chain of the HI-TCR. In certain embodiments, the TCR constant domain locus can be TRAC, TRBC1, TRBC2, TRGC1, or TRGC2. In certain embodiments, the HI-TCR expression cassette comprises an 20 extracellular antigen-binding domain that is integrated in the first exon of a TRAC locus, so that the extracellular antigen-binding domain and a TRAC peptide are comprised in a first antigen binding chain of the HI-TCR. In certain embodiments, the HI-TCR expression cassette further comprises a second antigen binding chain, which optionally comprises an extracellular antigen-binding domain and a TRBC peptide. In certain 25 embodiments, the HI-TCR expression cassette comprises an extracellular antigen-binding domain that is integrated in the first exon of a TRBC locus, so that the extracellular antigen-binding domain and a TRBC peptide are comprised in a first antigen binding chain of the HI-TCR. In certain embodiments, the HI-TCR expression cassette further comprises a second antigen binding chain, which optionally comprises an extracellular 30 antigen-binding domain and a TRAC peptide. In certain embodiments, the expression cassette comprises elements that create polycistronic expression cassette, e.g., a cleavable peptide, e.g., a 2A peptide. In certain embodiments, the recombinant TCR is expressed from an expression cassette placed in an endogenous TRAC locus and / or a TRBC locus of an 2024220100   26 Sep 2024 immunoresponsive cell. In certain embodiments, the placement of the recombinant TCR expression cassette disrupts or abolishes the endogenous expression of a TCR comprising a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell. In certain embodiments, the placement of the recombinant TCR expression cassette prevents 5 or eliminates mispairing between the recombinant TCR and a native TCR a chain and / or a native TCR P chain in the immunoresponsive cell. Any suitable genetic editing methods and systems can be used to modify an endogenous T cell receptor locus. The genome editing methods disclosed in Section 4 can be used to modify the endogenous T cell receptor locus. In certain embodiments, a 10 CRISPR system is used to modify T cell receptor locus. In certain embodiments, the CRISPR system targets exon 1 of a human TRAC locus. In certain embodiments, the CRISPR system comprises a guide RNA (gRNA) that targets exon 1 of a human TRAC locus. In certain embodiments, a zinc-finger nuclease is used to modify an endogenous T 15 cell receptor locus. In certain embodiments, a TALEN system is used to modify an endogenous T cell receptor locus. In certain embodiments, when one endogenous T cell receptor locus in a cell is modified to express one or more antigen binding chain of an HI-TCR, one or more other endogenous T cell receptor locus in the cell are modified to eliminate the endogenous 20 expression of the endogenous TCR chain. In certain embodiments, the one or more other endogenous T cell receptor locus are further modified to express a gene of interest, e.g., an anti-tumor cytokine (e.g., IL-2, IL-12, TNFa, and INFy), a co-stimulatory molecule ligand (e.g., 4-1BBL), a tracking gene (e.g., eGFP) or a suicide gene. 6.2. Modifying Gene Expression through Genome Editing of a Promoter or a 25 Transcription Terminator In certain non-limiting embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is driven by an endogenous promoter / enhancer of the genomic locus. In certain embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is driven by a 30 modified promoter / enhancer introduced to the genomic locus. Any targeted genome editing methods can be used to modify the promoter / enhancer region of a targeted genomic locus, and thereby enhancing or modifying the expression of an HI-TCR in an immunoresponsive cell. In certain embodiments, the modification comprises replacement of an endogenous promoter with a constitutive promoter or an inducible promoter, or 2024220100   26 Sep 2024 insertion of a constitutive promoter or inducible promoter to the promoter region of a targeted genomic locus. In certain embodiments, a constitutive promoter is positioned on a targeted genomic locus to drive gene expression of the HI-TCR. Eligible constitutive promoters include, but are not limited to, a CMV promoter, an EFla promoter, a SV40 5 promoter, a PGK1 promoter, a Ube promoter, a beta-actin promoter, and a CAG promoter. Alternatively or additionally, a conditional or inducable promoter is positioned on a targeted genomic locus to drive gene expression of the HI-TCR. Non-limiting examples of conditional promoters include a tetracycline response element (TRE) promoter and an estrogen response element (ERE) promoter. In addition, enhancer 0 elements can be placed in regions other than the promoter region. In certain non-limiting embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is regulated by an endogenous transcription terminator of the genomic locus. In certain embodiments, the expression of an HI-TCR expression cassette integrated into a targeted genomic locus is regulated by a 15 modified transcription terminator introduced to the genomic locus. Any targeted genome editing methods can be used to modify the transcription terminator region of a targeted genomic locus, and thereby modifying the expression of an HI-TCR in an immunoresponsive cell. In certain embodiments, the modification comprises replacement of an endogenous transcription terminator with an alternative transcription terminator, or 20 insertion of an alternative transcription terminator to the transcription terminator region of a targeted genomic locus. In certain embodiments, the alternative transcription terminator comprises a 3’UTR region or a ploy A region of a gene. In certain embodiments, the alternative transcription terminator is endogenous. In certain embodiments, the alternative transcription terminator is exogenous. In certain embodiments, alternative 25 transcription terminators include, but are not limited to, a TK transcription terminator, a GCSF transcription terminator, a TCRA transcription terminator, an HBB transcription terminator, a bovine growth hormone transcription terminator, an SV40 transcription terminator and a P2A element. Any targeted genome editing methods can be used to modify the 30 promoter / enhancer region and / or the transcription terminator region of a targeted genomic locus. In certain embodiments, a CRISPR system is used to modify the promoter / enhancer region and / or the transcription terminator region of a targeted genomic locus. In certain embodiments, zinc-finger nucleases are used to modify the promoter / enhancer region and / or the transcription terminator region of a targeted genomic 2024220100   26 Sep 2024 locus. In certain embodiments, a TALEN system is used to modify the promoter / enhancer region and / or the transcription terminator region of a targeted genomic locus. Methods for delivering the genome editing agents / systems can vary depending on the need. In certain embodiments, the components of a selected genome editing method 5 are delivered as DNA constructs in one or more plasmids. In certain embodiments, the components are delivered via viral vectors. Common delivery methods include but is not limited to, electroporation, microinjection, gene gun, impalefection, hydrostatic pressure, continuous infusion, sonication, magnetofection, adeno-associated viruses, envelope protein pseudotyping of viral vectors, replication-competent vectors cis and trans-acting 10 elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic Nanoparticles, and cell-penetrating peptides). Modification can be made anywhere within a targeted genomic locus, or anywhere that can impact gene expression of a targeted genomic locus. In certain embodiments, the 15 modification occurs upstream of the transcriptional start site of a targeted genomic locus. In certain embodiments, the modification occurs between the transcriptional start site and the protein coding region of a targeted genomic locus. In certain embodiments, the modification occurs downstream of the protein coding region of a targeted genomic locus. In certain embodiments, the modification occurs upstream of the transcriptional start site 20 of a targeted genomic locus, wherein the modification produces a new transcriptional start site. 7. Polypeptides and Analogs Also included in the presently disclosed subject matter are polypeptides (e.g., CD19, CD8, CD28, CD3i; CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, 25 ICAM-1, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, CD3y, CD38, CD3s and CTLA-4) or fragments thereof that are modified in ways that enhance their anti-neoplastic activity when expressed in an immunoresponsive cell. The presently disclosed subject matter provides methods for optimizing an amino acid sequence or nucleic acid sequence by producing an alteration in the sequence. Such alterations may include certain mutations, 30 deletions, insertions, or post-translational modifications. The presently disclosed subject matter further includes analogs of any naturally-occurring polypeptide disclosed herein (including, but not limited to, CD19, CD8, CD28, CD3i; CD40, 4-1BB, OX40, CD84, CD 166, CD8a, CD8b, ICOS, ICAM-1, TRAC, TRBC1, TRBC2, TRGC1, TRGC2, CD3y, CD36, CD3s and CTLA-4). Analogs can differ from a naturally-occurring 2024220100   26 Sep 2024 polypeptide disclosed herein by amino acid sequence differences, by post-translational modifications, or by both. Analogs can exhibit at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more homologous to all or part of a naturally-occurring amino, acid 5 sequence of the presently disclosed subject matter. The length of sequence comparison is at least 5, 10, 15 or 20 amino acid residues, e.g., at least 25, 50, or 75 amino acid residues, or more than 100 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3 and e'100 indicating a closely related sequence. Modifications include in vivo and in 0 vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting 15 from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amina acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., P or y 20 amino acids. In addition to full-length polypeptides, the presently disclosed subject matter also provides fragments of any one of the polypeptides or peptide domains disclosed herein. As used herein, the term “a fragment” means at least 5, 10, 13, or 15 amino acids. In certain embodiments, a fragment comprises at least 20 contiguous amino acids, at least 30 25 contiguous amino acids, or at least 50 contiguous amino acids. In certain embodiments, a fragment comprises at least 60 to 80, 100, 200, 300 or more contiguous amino acids. Fragments can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by 30 alternative mRNA splicing or alternative protein processing events). Non-protein analogs have a chemical structure designed to mimic the functional activity of a protein disclosed herein. Such analogs may exceed the physiological activity of the original polypeptide. Methods of analog design are well known in the art, and 2024220100   26 Sep 2024 synthesis of analogs can be carried out according to such methods by modifying the chemical structures such that the resultant analogs increase the anti-neoplastic activity of the original polypeptide when expressed in an immunoresponsive cell. These chemical modifications include, but are not limited to, substituting alternative R groups and varying 5 the degree of saturation at specific carbon atoms of a reference polypeptide. In certain embodiments, the protein analogs are relatively resistant to in vivo degradation, resulting in a more prolonged therapeutic effect upon administration. Assays for measuring functional activity include, but are not limited to, those described in the Examples below. 8. Administration 10          Compositions comprising the presently disclosed immunoresponsive cells can be provided systemically or directly to a subject for inducing and / or enhancing an immune response to an antigen and / or treating and / or preventing a neoplasia, pathogen infection, or infectious disease. In certain embodiments, the presently disclosed immunoresponsive cells or compositions comprising thereof are directly injected into an organ of interest 15   (e.g., an organ affected by a neoplasia). Alternatively, the presently disclosed immunoresponsive cells or compositions comprising thereof are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the tumor vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of the cells or compositions to increase production of T cells, NKT 20 cells, or CTL cells in vitro or in vivo. The presently disclosed immunoresponsive cells can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). Usually, at least about 1 * 105 cells 25 will be administered, eventually reaching about 1 * 1010 or more. The presently disclosed immunoresponsive cells can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of the presently disclosed immunoresponsive cells in a population using various well-known methods, such as fluorescence activated cell sorting (FACS). Suitable ranges of purity in populations comprising the presently 30 disclosed immunoresponsive cells are about 50% to about 55%, about 5% to about 60%, and about 65% to about 70%. In certain embodiments, the purity is about 70% to about 75%, about 75% to about 80%, or about 80% to about 85%. In certain embodiments, the purity is about 85% to about 90%, about 90% to about 95%, and about 95% to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in 2024220100   26 Sep 2024 purity may require an increase in dosage). The cells can be introduced by injection, catheter, or the like. The presently disclosed compositions can be pharmaceutical compositions comprising the presently disclosed immunoresponsive cells or their progenitors and a 5 pharmaceutically acceptable carrier. Administration can be autologous or heterologous. For example, immunoresponsive cells, or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic 0 injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising a presently disclosed immunoresponsive cell), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion). 9. Formulations 15          Compositions comprising the presently disclosed immunoresponsive cells can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat 20 more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid 25 polyethylene glycol, and the like) and suitable mixtures thereof. Sterile injectable solutions can be prepared by incorporating the genetically modified immunoresponsive cells in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological 30 saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON’S 2024220100   26 Sep 2024 PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation. Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be 5 added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or 10 additive used would have to be compatible with the genetically modified immunoresponsive cells or their progenitors. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as 15 dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride can be particularly for buffers containing sodium ions. Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. For example, methylcellulose is is readily and economically available and is easy to work with. Other suitable thickening 20 agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid 25 dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form). The quantity of cells to be administered will vary for the subject being treated. In a one embodiment, between about 104 and about 1010, between about 105 and about 109, or between about 106 and about 108 of the presently disclosed immunoresponsive cells are 30 administered to a human subject. More effective cells may be administered in even smaller numbers. In certain embodiments, at least about 1 * 108, about 2* 108, about 3*108, about 4*108, or about 5*108 of the presently disclosed immunoresponsive cells are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, 2024220100   26 Sep 2024 age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions and to be administered in methods. 5 Typically, any additives (in addition to the active cell(s) and / or agent(s)) are present in an amount of 0.001 to 50% (weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as about 0.0001 to about 5 wt %, about 0.0001 to about 1 wt %, about 0.0001 to about 0.05 wt% or about 0.001 to about 20 wt %, about 0.01 to about 10 wt %, or about 0.05 to about 5 wt %. For 0 any composition to be administered to an animal or human, the followings can be determined: toxicity such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the 15 knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation. 10. Methods of Treatment The presently disclosed subject matter provides methods for inducing and / or increasing an immune response in a subject in need thereof. The presently disclosed 20 immunoresponsive cells and compositions comprising thereof can be used for treating and / or preventing a neoplasia in a subject. The presently disclosed immunoresponsive cells and compositions comprising thereof can be used for prolonging the survival of a subject suffering from a neoplasia. The presently disclosed immunoresponsive cells and compositions comprising thereof can also be used for treating and / or preventing a 25 pathogen infection or other infectious disease in a subject, such as an immunocompromised human subject. Such methods comprise administering the presently disclosed immunoresponsive cells in an amount effective or a composition (e.g., pharmaceutical composition) comprising thereof to achieve the desired effect, be it palliation of an existing condition or prevention of recurrence. For treatment, the amount 30 administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion. In certain embodiments, immunoresponsive cells comprising a HI-TCR disclosed herein can be used to treat a subject having tumor cells with a low expression level of a 2024220100   26 Sep 2024 surface antigen, .e.g, from a relapse of a disease, wherein the subject received treatment which leads to residual tumor cells. In certain embodiments, the tumor cells have low density of a target molecule on the surface of the tumor cells In certain embodiments, a target molecule having a low density on the cell surface has a density of less than about 5   5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, a target molecule having a low density on 10 the cell surface has a density of less than about 2,000 molecules per cell. In certain embodiments, a target molecule having a low density on the cell surface has a density of less than about 1,500 molecules per cell. In certain embodiments, a target molecule having a low density on the cell surface has a density of less than about 1,000 molecules per cell. In certain embodiments, a target molecule having a low density on the cell 15   surface has a density of between about 4,000 molecules per cell and about 2,000 molecules per cell, between about 2,000 molecules per cell and about 1,000 molecules per cell, between about 1,500 molecules per cell and about 1,000 molecules per cell, between about 2,000 molecules per cell and about 500 molecules per cell, between about 1,000 molecules per cell and about 200 molecules per cell, or between about 1,000 molecules 20 per cell and about 100 molecules per cell. In certain embodiments, immunoresponsive cells comprising a HI-TCR disclosed herein can be used to treat a subject having a relapse of a disease, wherein the subject received immunoresponsive cells (e.g., T cells) comprising a CAR comprising an intracellular signaling domain that comprises a costimulatory signaling domain comprising a 4-1BB polypeptide (e.g., a 4-lBBz CAR). In 25 certain embodiments, the tumor cells have a low density of a tumor specific antigen on the surface of the tumor cells. In certain embodiments, the disease is CD19+ ALL. In certain embodiments, the tumor cells have a low density of CD 19 on the tumor cells. Such methods comprise administering the presently disclosed immunoresponsive cells in an amount effective or a composition (e.g., pharmaceutical composition) comprising 30 thereof to achieve the desired effect, alleviation of an existing condition or prevention of recurrence. An “effective amount” (or, “therapeutically effective amount”) is an amount sufficient to effect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an 2024220100   26 Sep 2024 effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into 5 account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the immunoresponsive cells administered. For adoptive immunotherapy using antigen-specific T cells, cell doses in the range 0   of about 106-1010 (e.g., about 109) are typically infused. Upon administration of the presently disclosed cells into the host and subsequent differentiation, T cells are induced that are specifically directed against the specific antigen. The modified cells can be administered by any method known in the art including, but not limited to, intravenous, subcutaneous, intranodal, intratumoral, intrathecal, intrapleural, intraperitoneal and 15   directly to the thymus. The presently disclosed subject matter provides methods for treating and / or preventing a neoplasia in a subject. The method can comprise administering an effective amount of the presently disclosed immunoresponsive cells or a composition comprising thereof to a subject having a neoplasia. 20          Non-limiting examples of neoplasia include blood cancers (e.g. leukemias, lymphomas, and myelomas), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas 25 (including prostate and small cell lung cancer). Suitable carcinomas further include any known in the field of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, 30 angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing’s tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, 2024220100   26 Sep 2024 papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, 5 oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular 10 and diffuse) plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas. In certain embodiments, the neoplasm is selected from the group consisting of blood cancers (e.g. leukemias, lymphomas, and myelomas), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin 15 cancer, stomach cancer, glioblastoma, and throat cancer. In certain embodiments, the presently disclosed immunoresponsive cells and compositions comprising thereof can be used for treating and / or preventing blood cancers (e.g., leukemias, lymphomas, and myelomas) or ovarian cancer, which are not amenable to conventional therapeutic interventions. In certain embodiments, the neoplasm is a solid tumor. 20          The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and / or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence. 25           Suitable human subjects for therapy typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with “advanced disease” or “high tumor burden” are those who bear a clinically measurable tumor. A clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram or X-ray; positive biochemical or histopathologic markers on 30 their own are insufficient to identify this population). A pharmaceutical composition is administered to these subjects to elicit an anti-tumor response, with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor. 2024220100   26 Sep 2024 A second group of suitable subjects is known in the art as the “adjuvant group.” These are individuals who have had a history of neoplasia, but have been responsive to another mode of therapy. The prior therapy can have included, but is not restricted to, surgical resection, radiotherapy, and traditional chemotherapy. As a result, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases. This group can be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment. These features are known in the clinical arts, and are suitably defined for each different neoplasia. Features typical of high-risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes. Another group have a genetic predisposition to neoplasia but have not yet evidenced clinical signs of neoplasia. For instance, women testing positive for a genetic mutation associated with breast cancer, but still of childbearing age, can wish to receive one or more of the immunoresponsive cells described herein in treatment prophylactically to prevent the occurrence of neoplasia until it is suitable to perform preventive surgery. Additionally, the presently disclosed subject matter provides methods for treating and / or preventing a pathogen infection (e.g., viral infection, bacterial infection, fungal infection, parasite infection, or protozoal infection) in a subject, e.g., in an immunocompromised subject. The method can comprise administering an effective amount of the presently disclosed immunoresponsive cells or a composition comprising thereof to a subject having a pathogen infection. Exemplary viral infections susceptible to treatment include, but are not limited to, Cytomegalovirus (CMV), Epstein Barr Virus (EBV), Human Immunodeficiency Virus (HIV), and influenza virus infections. Further modification can be introduced to the presently disclosed immunoresponsive cells (e.g., T cells) to avert or minimize the risks of immunological complications (known as “malignant T-cell transformation”), e.g., graft versus-host disease (GvHD), or when healthy tissues express the same target antigens as the tumor cells, leading to outcomes similar to GvHD. A potential solution to this problem is engineering a suicide gene into the presently disclosed immunoresponsive cells. Suitable suicide genes include, but are not limited to, Herpes simplex virus thymidine kinase (hsv-tk), inducible Caspase 9 Suicide gene (iCasp-9), and a truncated human epidermal growth factor receptor (EGFRt) polypeptide. In certain embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can enable T cell elimination by 2024220100   26 Sep 2024 administering anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently joined to the upstream of the antigen-recognizing receptor of a presently disclosed CAR. The suicide gene can be included within the vector comprising nucleic acids encoding a presently disclosed CAR. In this way, administration of a prodrug 5 designed to activate the suicide gene (e.g., a prodrug (e.g., API903 that can activate iCasp-9) during malignant T-cell transformation (e.g., GVHD) triggers apoptosis in the suicide gene-activated CAR-expressing T cells. The incorporation of a suicide gene into the a presently disclosed CAR gives an added level of safety with the ability to eliminate the majority of CAR T cells within a very short time period. A presently disclosed 10 immunoresponsive cell (e.g., a T cell) incorporated with a suicide gene can be preemptively eliminated at a given timepoint post CAR T cell infusion, or eradicated at the earliest signs of toxicity. 11. Kits The presently disclosed subject matter provides kits for inducing and / or enhancing 15 an immune response and / or treating and / or preventing a neoplasia or a pathogen infection in a subject. In certain embodiments, the kit comprises an effective amount of presently disclosed immunoresponsive cells or a pharmaceutical composition comprising thereof. In certain embodiments, the kit comprises a sterile container; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable 20 container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. In certain non-limiting embodiments, the kit includes an isolated nucleic acid molecule encoding an HI-TCR disclosed herein which is directed toward an antigen of interest in expessible form, which may optionally be comprised in one or more vectors. 25          If desired, the immunoresponsive cells and / or nucleic acid molecules are provided together with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing a neoplasia or pathogen or immune disorder. The instructions generally include information about the use of the composition for the treatment and / or prevention of neoplasia or a pathogen infection. In certain 30 embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neoplasia, pathogen infection, or immune disorder or symptoms thereof; precautions; warnings; indications; counter-indications; over-dosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed 2024220100   26 Sep 2024 directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. EXAMPLES The practice of the present disclosure employs, unless otherwise indicated, 5 conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); 0 “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); ’’Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides 15 disclosed herein, and, as such, may be considered in making and practicing the the presently disclosed subject matter. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the presently 20 disclosed cells and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Example 1 - Tarsetins T cells with endosenous non-HLA restricted T cell receptor / CD3 complexes Introduction 25           Specific modifications were made at a TCR locus designed to alter the antigen specificity of human T cells without disrupting or bypassing their CD3 complex, which physiologically controls and regulates T cell activation. In this approach, the T cell lost its endogenous T cell receptor and acquired the ability to recognize antigen independent of HLA, making use of its CD3 complex, which is otherwise bypassed when using CARs. 30 This was accomplished through the targeted disruption of the endogenous TCR combined with the introduction of antigen binding domains into the TRAC locus in such a way as to preserve all components of the natural CD3 complex in their native structure. The recombinant TCR-like molecule (i.e., HI-TCR, FvTCR or HIT-CAR) can harbor variable domains derived from immunoglobulin genes, or alternative ligands, to direct antigen 2024220100   26 Sep 2024 recognition. The recombined receptor signals via the CD3 complex, which can either be intact (i.e., in its physiological composition) or augmented through the non-covalent incorporation of costimulatory domains. Results 5          A novel strategy for one-step generation of TCR and TCR-like T cells was developed by integrating a TCR or TCR-like gene with a distinct variable domain for a predetermined antigen. This resulted in the expression of the new TCR / TCR-like gene under the control of the endogenous TCR promoter (alpha, beta or both) with concomitant disruption of surface expression of the endogenous TCR. 10          A strategy for one-step generation of universal CAR T cells were developed by targeting the integration of a promoter-less CAR gene cassette in the TCR alpha constant chain (TRAC) first exon. This resulted in CAR expression under the control of the endogenous TCR alpha promoter with concomitant disruption of the TCR alpha gene expression, leading to lack of TCR expression at the cell surface. CAR gene targeting at 15 the TRAC locus was accomplished through homologous recombination (HR) by using a site-specific nuclease (e.g. CRISPR / Cas9) and an AAV donor template. Using HR-based gene targeting, the present strategy permitted the generation of T cells with a unique specificity, which was encoded by a chimeric TCR receptor containing a specific antigenbinding domain. The antigen-binding domain was derived from either an 20 Immunoglobulin (i.e. Fv fragment), a ligand for a cell-surface receptor or a TCR (aP or y5). The hybrid Immunoglobulin-TCR chimeric antigen receptor (i.e., HIT-CAR, FvTCR or HI-TCR) or the ySTCR allowed the T cell to recognize a target cell in an HLA-independent manner. Inserting a new aPTCR could be accomplished in the same way but would result in HLA-restricted antigen recognition. 25          For example, Figure 1A shows schematic representation of the T Cell Receptor (TCR), the B Cell Receptor (BCR), a Chimeric Antigen Receptor (CAR) and the HLA-Independent TCR-based Chimeric Antigen Receptor (i.e., HIT-CAR, FvTCR or HI-TCR). CRISPR / Cas9-targeted integration of the three receptors into the TRAC locus is shown in Figure IB. The engineered HIT-CAR targets CD19. Cell surface expression of 30 HIT-CAR (i.e., HI-TCR) from TRAC locus is show in Figure IC. Cytolitic effects and proliferation of HIT-CAR (i.e., HI-TCR or FvTCR) T cells are shown in Figures ID and IE respectively. Conditions were established to yield up to 65% of HIT-CAR (i.e., HI-TCR or FvTCR) T cells combining target gene disruption and HIT-CAR (i.e., HI-TCR or FvTCR) 2024220100   26 Sep 2024 targeted insertion in a single step as shown in Figure 2A. These T cells exhibited in vitro and in vivo tumor lysis activity similar to the previously characterized TRAC-CAR T cells expressing the 1928z CAR as shown in Figure 2B. In addition, antigen interaction induced down-regulation of the HIT-CAR (i.e., HI-TCR or FvTCR) T cells, which was 5 dependent of the number of antigen-dependent stimulations. As endogenous TCR expression is eliminated at the cell surface, these HIT-CAR (i.e., HI-TCR or FvTCR) T cells were useful for the development of off-the-shelf immunotherapy. Targets other than CD 19 can be employed for immunotherapy. Schematic representation of the NYESO TCR genes integrated into the TCR alpha or beta chain is 0 shown in Figure 3 A. Cell surface expressions of TRAC-NYESO-TCR and TRBC-NYESO-TCR (i.e., HI-TCRs or FvTCRs) are show in Figure 3B. Representative TCR-V-beta-1 flow cytometry plots 4 days after TRAC or TRBC targeting are shown in Figure 3B. Cytotoxic activity of the engineered T cells is shown in Figure 3C. Schematic representation of an alternative design of co-targeting into both the TCR alpha and the 15   TCR beta is shown in Figure 3D, where a NYESO-HI-TCR was placed in the TRAC locus and 4-1BBL expression cassette was placed in TRBC locus. Cell surface expressions of the TRAC-NYESO-TCR and TRBC-4-1BBL are shown in Figure 3E. A variant approach was also provided in which a costimulatory domain is non-covalently inserted into the CD3 complex, which provides supraphysiological antigen 20 sensing and activation. This is achieved by fusing the costimulatory domain to one or both TCR chains. Moreover, two costimulatory dosages are possible, by ether fusing the costimulatory domain to one or both modified TCR chains (i.e., antigen binding chains). Moreover, HI-TCR exhibited greater sensitivity in comparison to CARs. Human T cells edited to replace endogenous TCR with HI-TCR acquired the ability to engage 25 lower antigen densities and kill such cells. For example, Table 2 shows in vitro cytotoxic activity of HI-TCR cells. HI-TCR or CD19-CAR was introduced at the endogenous TRAC locus of human peripheral T cells via CRISPR / Cas9-mediated gene editing and AAV6 donor vectors. Five days post-gene targeting, T cells (6, 30, or 150 thousands) were incubated with 250 thousands Nalm6 leukemia cells that express different CD 19 30 levels (from very low to high levels). Cells were incubated for 22h in 500-ul of X-Vivo medium containing serum (without IL2). Co-cultures were analyzed by FACS in the presence of counting beads to determine the total number of Nalm6 cells. Cytotoxic activity is shown as a percentage of Nalm6 killed. E / T: effector (T cells) to target (Nalm6) ratio. The data clearly demonstrate that the HI-TCR can detect lower levels of 2024220100   26 Sep 2024 CD 19 antigen than a CAR. This feature can be very useful for antigens with moderate or low expression such as CD22, BCMA, CCR1, CD70, etc., and can also be useful to treat relapse after CAR therapy for any antigen, as the relapse tumor cells frequently show reduced antigen densities on their surface. Table 2. Tumor Cell Killing Percem tage E / T HI-TCR CD19CAR CD19 levels 0.024 0 0 very low 22.53 16.32 low 29.19 17.18 medium 38.3 32 high 0.12 15.48 0.4 very low 51.46 53.31 low 54.14 63.58 medium 63.28 63.38 high 0.6 30.36 8.37 very low 68.32 77.2 low 77.01 79.38 medium 78.72 84.37 high Furthermore, a panel of exogenous and endogenous 3' unstranslated regions (3’UTR) were found as capable of regulate precise and predictable gene expression at the TCR locus. For example, Figure 4A shows schematic representation of the CD28z CAR 10 gene integrated into the TRAC locus. Poly A (black box) corresponds to the segment of the CAR cassette that was modified to test different viral and mammalian 3’ UTRs. Using TRAC-CAR T cells expressing the CD28z CAR as a model, certain 3’UTRs were shown to lower the CAR expression compared to the bovine growth hormone (bGH) polyA sequence (Figures 4B and 4C), which yielded CAR-T cells with impaired in vivo 15   cytotoxic activity (Figures 5 A and 5B). Certain other 3 UTRs, including the endogenous TRAC 3'UTR, were shown to increase CAR surface expression levels (Figures 4B and 4C), which produced CAR-T cells with improved in vivo cytotoxic activity (Figures 5A and 5B). These 3’UTRs can also be used to regulate precise and predictable gene expression of any HI-TCR disclosed herein. 2024220100   26 Sep 2024 The above-described genetic modifications at the TRAC locus permit the generation of T cells expressing optimal levels of HI-TCR that like the physiological TCR but unlike CARs, take advantage of the endogenous T cell activating machinery (the CD3 complex and downstream signaling elements). This approach can advance both 5 autologous and allogeneic T cell therapies. Another relevant area where TCR gene editing has relevant applications is T-iPS-derived T cells. T-iPS cells are pluripotent stem cells obtained through reprogramming of peripheral T lymphocytes. These T-iPS cells therefore contain a rearranged T-cell receptor, either an aP or a y6 TCR, which can be modified using gene-editing 0 technologies. T cells obtained from T-iPS cells through directed differentiation express the rearranged TCR, which can be detected and sequenced. Using this sequence, one can determine the precise location of the rearranged variable domain in the genome of T-iPS cells. Using nucleases that specifically target this rearranged variable domain and a donor DNA containing a TCR variable domain of known specificity, one can replace the 15 endogenous variable domain with the new one. This approach requires two steps of gene editing: one targeting the a (or y) chain, and the other the P (or 6) chain. The approach can be performed in a single step since only two alleles need to be modified. In addition, using a strategy similar as to the one described for primary human T cells, the TCR chains can be modified to express any HI-TCR disclose herein. 20 Example 2 CRISPR / Cas9-targeted integration of the three receptors into the TRAC locus is shown in Figure 1 A. The engineered HIT-CAR targets CD19. Cell surface expression of HIT-CAR (i.e., HI-TCR) from TRAC locus is show in Figures 6B and 6C. Cytolitic effects and proliferation of HIT-CAR (i.e., HI-TCR or FvTCR) T cells are shown in 25 Figures 7A,7B and 8A-8C. In particular, these data show that HIT T cells outperformed CAR-T cells at killing target cells expressing low antigen levels. Furthermore, as shown in Figures 9A-9D the HI-TCR T cells were engineered to co-express costimulatory ligands. In this specific experiment, the day after targeting the HI-TCR to the TRAC locus, the T cells were transferred with retroviral SFG vectors 30 coding for CD80, 41BBL or both. 5 days after expanding these cells ex vivo, 4e5 TRAC-CAR or TRAC-HI-TCR positive T cells were injected into NSG mice bearing NALM6 cells expressing very low level of CD 19. The TRAC-HI-TCR T cells we either expressing CD80, 41BBL, both ligand or none. Bioluminescence was used to assess the tumor burden every week and follow the survival of the mice. 2024220100   26 Sep 2024 It was observed that the TRAC-CAR T cells were not able to control the tumor with very low levels of CD19 and all the mice were terminally ill by day 30. At this low dose, the HI-TCR T cells initially controlled the tumor burden, however the mice relapsed by day 10 and were terminally ill by day 40. The addition of costimulatory ligands to the 5 HI-TCR T cells improved the anti-tumor activity with an optimal response when the HI-TCR T cells co-expressed CD80 and 4-1BBL. This improved activity resulted in a longterm control of NALM6 expressing very low levels of CD19. Figures 10A-10C further demonstrate that baseline TRAC-CAR expression can be controlled by distinct 3'UTR sequences without affecting cell surface replenishment 10 kinetic after antigen encounter. The TRAC-CAR T cells were engineered the same way as previously described, so that the all the constructs were under the transcriptional control of the TRAC endogenous promoter. It was observed that by modifying the 3’UTR (that includes polyA signal), the baseline expression level were modulated(Figure 10B). When these different TRAC-CAR T cells were cultivated on CD 19 expressing 15 tumor cells, a drop in the CAR cell surface expression followed by a replenishment was observed by flow cytometry (Figure 10 C). The different 3’UTR changed the baseline expression level but they retained the same replenishment kinetic and the final expression level was similar to the baseline. Furthermore, a HI-TCR targeting CD70 and a HI-TCR targeting CD22 were also 20 created. HI-TCR targeting an interested antigen can be created by sequencing an existing scFv or a Fab region of an existing antibody targeting the same antigen to obtain the extracellular antigen-binding domain. Embodiments of the presently disclosed subject matter 25           From the foregoing description, it will be apparent that variations and modifications may be made to the presently disclosed subject matter to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. The recitation of a listing of elements in any definition of a variable herein 30 includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. 2024220100   17 Sep 2026 All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. A reference herein to a patent document or other matter which is given as prior art is 5 not to be taken as admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Claims

1. A method of treating a tumor cell in a subject, the method comprising administering to the subject an effective amount of immunoresponsive cells, wherein the immunoresponsive cell comprises a recombinant T cell receptor (TCR) that comprises:i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; andii) a second antigen-binding chain comprising an antigen-binding fragment of a light chain variable region (VL) of the antibody;wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind to an antigen, andwherein the tumor cell expresses the antigen on its surface at a density of less than about 10,000 molecules per cell.

2. Use of an immunoresponsive cell for the manufacture of a medicament for treating a tumor cell in a subject, wherein the immunoresponsive cell comprises a recombinant T cell (TCR) that comprises:i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; andii) a second antigen-binding chain comprising an antigen-binding fragment of a light chain variable region (VL) of the antibody;wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind to an antigen, andwherein the tumor cell expresses the antigen on its surface at a density of less than about 10,000 molecules per cell.

3. A method of treating a tumor cell in a subject, the method comprising administering to the subject an effective amount of immunoresponsive cells, wherein the immunoresponsive cell comprises a recombinant T cell receptor (TCR) that comprises:i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; and2024220100   17 Sep 2026ii) a second antigen-binding chain comprising an antigen-binding fragment of a light chain variable region (VL) of the antibody;wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind to an antigen with a dissociation constant (Kd) of about 1x10-8 M or less, and the tumor cell expresses the antigen.

4. Use of an immunoresponsive cell for the manufacture of a medicament for treating a tumor cell in a subject, wherein the immunoresponsive cell comprises a recombinant T cell (TCR) that comprises:i) a first antigen-binding chain comprising an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; andii) a second antigen-binding chain comprising an antigen-binding fragment of a light chain variable region (VL) of the antibody;wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, and the first and the second antigen-binding chains bind to an antigen with a dissociation constant (KD) of about 1x10-8 M or less, and the tumor cell expresses the antigen.

5. The method of claim 1 or 3, or use of claim 2 or 4, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous.

6. The method of claim 1 or 3, or use of claim 2 or 4, wherein the immunoresponsive cell is a cell of lymphoid lineage or a pluripotent stem cell from which a cell of lymphoid lineage may be differentiated.

7. The method of claim 1 or 3, or use of claim 2 or 4, wherein the immunoresponsive cell is a B cell, a T cell, a Natural Killer (NK) cell, a pluripotent stem cell from which a lymphoid cell may be differentiated, or a combination thereof.

8. The method of any one of claims 3, or 5 to 7, or use of any one of claims 4 to 7, wherein the tumor cell expresses the antigen on its surface at a density of less than about 10,000 molecules per cell.2024220100   17 Sep 20269. The method of any one of claims 1, 3, or 5 to 8, or use of any one of claims 2, or 4 to 8, wherein the antigen is selected from the group consisting of CD19, MUC16, MUC1, CAIX, CEA, CD8, CD7, CD10, CD20, CD22, CD30, CLL1, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, erb-B2,3,4, FBP, Fetal acetylcholine receptor, folate receptor-a, GD2, GD3, HER-2, hTERT, IL-13R-a2, K-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, Mesothelin, ERBB2, MAGEA3, p53, MART1, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, NY-ESO-1, oncofetal antigen (h5T4), PSCA, PSMA, ROR1, TAG-72, VEGF-R2, WT-1, BCMA, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME, and ERBB.

10. The method or use of claim 8, wherein antigen is selected from the group consisting of CD22, BCMA, CCR1, and CD70.

11. The method of any one of claims 1, 3, or 5 to 10, or use of any one of claims 2, or 4 to 10, wherein the recombinant TCR is expressed by a transgene that is integrated at an endogenous gene locus of the immunoresponsive cell.

12. The method or use of claim 11, wherein the endogenous gene locus is a CD38 locus, a CD3s locus, a CD247 locus, a B2M locus, a TRAC locus, a TRBC locus, a TRDC locus and / or a TRGC locus.

13. The method or use of claim 11 or claim 12, wherein the endogenous expression of a TCR comprising a native TCR a chain and / or a native TCR 0 chain is disrupted or abolished in the immunoresponsive cell, thereby preventing or eliminating mispairing between the recombinant TCR and a native TCR a chain and / or a native TCR 0 chain in the immunoresponsive cell.

14. The method or use of any one of claims 11 to 13, wherein the endogenous gene locus is a first endogenous TCR locus, and a second endogenous TCR locus that is different from the first endogenous TCR locus is modified to eliminate the expression of an endogenous TCR chain encoded by the second endogenous TCR locus.2024220100   17 Sep 202615. The method of any one of claims 1, 3, or 5 to 14, or use of any one of claims 2, or 4 to 14, wherein the tumor cell is a cancer cell.

16. The method of any one of claims 1, 3, or 5 to 15, or use of any one of claims 2, or 4 to 15, wherein the tumor cell is a relapsed tumor cell.

17. The method of any one of claims 1, 3, or 5 to 16, or use of any one of claims 2, or 4 to 16, wherein the tumor cell is a solid tumor cell.

18. The method of any one of claims 1, 3, or 5 to 16, or use of any one of claim 2, or 4 to 16, wherein the tumor cell is a hematologic malignancy cell.

19. The method of any one of claims 1, 3, or 5 to 16, or use of any one of claims 2, or 4 to 16, wherein the tumor cell is a blood cancer cell.

20. The method of any one of claims 1, 3, or 5 to 16, or use of any one of claims 2, or 4 to 16, wherein the tumor cell is selected from the group consisting of B cell leukemia cells, multiple myeloma cells, acute lymphoblastic leukemia (ALL) cells, chronic lymphocytic leukemia (CLL) cells, Hodgkin's lymphoma cells, non-Hodgkin's lymphoma cells, Acute myeloid leukemia (AML) cells, and adenocarcinoma cells.

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