Enhanced antigen-recognizing receptors

Abstract

The presently disclosed subject matter provides compositions and methods for targeting immune responses toward tumor antigen-bearing cells. It relates to compositions, e.g., modified immunoresponsive cells, comprising an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) comprising a pTCR polypeptide. These cells have improved activity and / or efficiency.

Description

[0001] ENHANCED ANTIGEN-RECOGNIZING RECEPTORS

[0002] CROSS-REFERENCES TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority to U. S. Patent Provisional Application No.

[0004] 63 / 714,713, filed October 31, 2024, the content of which is incorporated herein by reference in its entirety, and to which priority is claimed.

[0005] SEQUENCE LISTING

[0006] The present specification makes reference to a Sequence Listing (submitted as an.xml file named 0727341847_ST26. The XML file was generated on October 30, 2025, and is 213,787 bytes in size. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0007] INTRODUCTION

[0008] The presently disclosed subject matter provides compositions and methods for targeting immune responses toward tumor antigen-bearing cells. It relates to compositions, e.g., modified immunoresponsive cells, comprising an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) comprising a pTCR polypeptide.

[0009] BACKGROUND OF THE INVENTION

[0010] Cell-based immunotherapy is a therapy with curative potential for the treatment of cancer. T cells and other immune cells may be modified to target tumor antigens through the introduction of genetic material coding for natural or modified T cell receptors (TCR) or synthetic receptors for antigen, termed Chimeric Antigen Receptors (CARs), specific to selected antigens. Patient-engineered CAR T cells have demonstrated remarkable efficacy against a range of liquid and solid malignancies. However, treatment failure and relapses occur in a large fraction of patients. Therefore, there remain needs for improved immunotherapy.

[0011] SUMMARY OF THE INVENTION

[0012] In certain non-limiting embodiments, the presently disclosed subject matter relates to an antigen-recognizing receptor including an extracellular antigen-binding domain that binds to an antigen, a transmembrane domain, and an intracellular domain, wherein the intracellular domain includes a pre-T cell receptor (pTCR) polypeptide.

[0013] In certain embodiments, the pTCR polypeptide includes a pTCRa polypeptide or a fragment thereof. In certain embodiments, the pTCR polypeptide includes an intracellular domain of a pTCRa polypeptide or a fragment thereof. In certain embodiments, the pTCR polypeptide includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the pTCR polypeptide includes the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the pTCR polypeptide includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the pTCR polypeptide includes the amino acid sequence set forth in SEQ ID NO: 18.

[0014] In certain embodiments, the antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is selected from CD 19, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein 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 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-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), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, Integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB. In certain embodiments, the tumor antigen is CD 19 or CD70.

[0015] In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or an HLA-independent T cell receptor (HIT).

[0016] In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). In certain embodiments, the intracellular signaling domain of the CAR includes a CD3(^ polypeptide. In certain embodiments, the CD3(^ polypeptide is a native CD3(^ polypeptide or a modified CD3(^ polypeptide. In certain embodiments, the native CD3(^ polypeptide includes the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the modified CD3(^ polypeptide includes a native IT AMI, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the modified CD3(^ polypeptide includes the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain of the CAR further includes at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region includes at least an intracellular domain of a co-stimulatory molecule or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, CD27, CD40, CD154, CD97, CDlla / CD18, ICOS, DAP- 10, CD2, CD 150, CD226, and NKG2D. In certain embodiments, the costimulatory molecule includes a CD28 polypeptide. In certain embodiments, the costimulatory molecule includes amino acids 180 to 220 of SEQ ID NO: 15. In certain embodiments, the CAR includes a transmembrane domain.

[0017] In certain embodiments, the antigen-recognizing receptor is a chimeric costimulatory receptor (CCR). In certain embodiments, the intracellular domain of the CCR does not alone deliver an activation signal to the cell. In certain embodiments, the intracellular domain of the CCR further includes at least an intracellular domain of a co-stimulatory molecule or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, CD27, CD40, CD154, CD97, CDlla / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0018] In certain embodiments, the antigen-recognizing receptor is HLA-independent T cell receptor (HIT). In certain embodiments, the HIT includes (i) a first antigen-binding chain including an antigen-binding fragment of a heavy chain variable region (VH) of an antibody; and (ii) a second antigen-binding chain including an antigen-binding fragment of a light chain variable region (VL) of the antibody; wherein each of the first and second antigen-binding chains includes the TRAC polypeptide or the TRBC polypeptide, and wherein the HIT binds to the antigen in an HLA-independent manner. In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the first and the second antigenbinding chains bind to the antigen with a dissociation constant (KD) of about 1 x IO’8M or less. In certain embodiments, the first and the second antigen-binding chains bind to the antigen with a dissociation constant (KD) of about 5 x 10'9M or less. In certain embodiments, the first antigenbinding chain includes an antigen-binding fragment of a VH of an antibody and a TRBC polypeptide, and the second antigen-binding chain includes an antigen-binding fragment of a VL of the antibody and a TRAC polypeptide. In certain embodiments, the first antigen-binding chain includes an antigen-binding fragment of a VH of an antibody and a TRAC polypeptide, and the second antigen-binding chain includes an antigen-binding fragment of a VL of the antibody and a TRBC polypeptide. In certain embodiments, the first and second antigen binding chains are capable of associating with a CD3(^ polypeptide. In certain embodiments, the first and second antigen binding chains, upon binding to the second antigen, are capable of activating the CD3(^ polypeptide.

[0019] In certain non-limiting embodiments, the presently disclosed subject matter relates to a nucleic acid encoding an antigen-recognizing receptor disclosed herein. In certain embodiments, the nucleic acid further includes a promoter that is operably linked to the antigen-recognizing receptor. In certain embodiments, the promoter is selected from the group consisting of an elongation factor (EF)-l promoter, a CMV promoter, a SV40 promoter, a PGK promoter, and a metallothionein promoter. In certain embodiments, the promoter is an inducible promoter selected from the group consisting of a NF AT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter.

[0020] In certain non-limiting embodiments, the presently disclosed subject matter relates to a vector or a lipid nanoparticle disclosed herein including the nucleic acid.

[0021] In certain non-limiting embodiments, the presently disclosed subject matter relates to a cell including an antigen-recognizing receptor, the nucleic acid, the vector, or the lipid nanoparticle disclosed herein. In certain embodiments, the antigen-recognizing receptor is recombinantly expressed. In certain embodiments, the antigen-recognizing receptor is expressed from a vector.

[0022] In certain embodiments, the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage. In certain embodiments, the cell of the lymphoid lineage is selected from T cells, B cells, Natural Killer (NK) cells, and dendritic cells. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is derived from an induced pluripotent stem cell. In certain embodiments, the T cell is a CD8+ T cell. In certain embodiments, the CD8+ T cell is CD4 independent. In certain embodiments, the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a yb T cell, a tumor-infiltrating lymphocyte (TIL), a virus-specific T cell (VST), a regulatory T cell, and a Natural Killer T (NKT) cell. In certain embodiments, the T cell is CD62L+. In certain embodiments, the T cell is CD45RA+. In certain embodiments, the T cell is CD45RA+ and CD62L+. In certain embodiments, the T cell is a tumor-infiltrating lymphocyte (TIL). In certain embodiments, the T cell is a virus-specific T cell (VST). In certain embodiments, the cell is an NK cell.

[0023] In certain embodiments, the antigen-recognizing receptor is integrated at a locus within the genome of the T cell. In certain embodiments, the locus is selected from the group consisting of a TRAC locus, a TRBC locus, a TRDC locus, and a TRGC locus. In certain embodiments, the locus is a TRAC locus or a TRBC locus. In certain embodiments, the locus is a TRAC locus.

[0024] In certain embodiments, the expression of the antigen-recognizing receptor is under the control of an endogenous promoter. In certain embodiments, the endogenous promoter is selected from the group consisting of an endogenous TRAC promoter, an endogenous TRBC promoter, an endogenous TRDC promoter, an endogenous TRGC promoter, and a combination thereof. In certain embodiments, the endogenous promoter is a TRAC promoter.

[0025] In certain embodiments, the cell further includes a gene disruption of a TRAC locus and / or a TRBC locus. In certain embodiments, the cell further includes a gene disruption of a CD70 locus. In certain embodiments, the cell further includes a gene disruption of a TRAC locus, a TRBC locus, and / or a CD70 locus. In certain embodiments, the cell further includes a gene modification of a TRAC gene and / or a TRBC gene.

[0026] In certain embodiments, the cell further includes at least one exogenous costimulatory ligand. In certain embodiments, the at least one exogenous co- stimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), CD40L. In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the at least one exogenous costimulatory ligand includes CD80. In certain embodiments, the at least one exogenous a costimulatory ligand includes 4-1BBL. In certain embodiments, the cell includes two exogenous costimulatory ligands. In certain embodiments, the at least two exogenous costimulatory ligands include CD80 and 4-1BBL. In certain embodiments, the at least two exogenous costimulatory ligands include the amino acid sequence set forth in SEQ ID NO: 67 and / or the amino acid sequence set forth in SEQ ID NO: 69.

[0027] In certain embodiments, the cell further includes a fusion polypeptide including: a) an extracellular domain and a transmembrane domain of a co-stimulatory ligand, and b) an intracellular domain of a first co-stimulatory molecule. In certain embodiments, the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof. In certain embodiments, the co-stimulatory ligand is CD80. In certain embodiments, the first co-stimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. In certain embodiments, the first co-stimulatory molecule is 4-1BB. In certain embodiments, the co-stimulatory ligand is CD80 and the first co-stimulatory molecule is 4- IBB. In certain embodiments, the fusion polypeptide includes the amino acid sequence set forth in SEQ ID NO: 71. In certain embodiments, the fusion polypeptide further includes an intracellular domain of a second costimulatory molecule. In certain embodiments, the second co-stimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. In certain embodiments, the second co-stimulatory molecule is CD28. In certain embodiments, the co-stimulatory ligand is CD80, the first co-stimulatory molecule is 4-1BB, and the second co-stimulatory molecule is CD28. In certain embodiments, the fusion polypeptide includes the amino acid sequence set forth in SEQ ID NO: 72.

[0028] In certain non-limiting embodiments, the presently disclosed subject matter relates to a cell including (a) a first antigen-recognizing receptor including a pre-T cell receptor (pTCR) polypeptide, and (b) a second antigen-recognizing receptor. In certain embodiments, (a) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a CAR; (b) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a CCR; (c) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a HIT; (d) the first antigen-recognizing receptor is a CAR and the second antigenrecognizing receptor is a recombinant TCR; (e) the first antigen-recognizing receptor is a CCR and the second antigen-recognizing receptor is a CAR; (f) the first antigen-recognizing receptor is a CCR and the second antigen-recognizing receptor is a HIT; or (g) the first antigen-recognizing receptor is a CCR and the second antigen-recognizing receptor is a recombinant TCR.

[0029] In certain embodiments, said cell is autologous. In certain embodiments, said cell is allogeneic.

[0030] In certain non-limiting embodiments, the presently disclosed subject matter relates to a composition including the cell, the nucleic acid, the vector, or the lipid nanoparticle disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further including a pharmaceutically acceptable excipient.

[0031] In certain non-limiting embodiments, the presently disclosed subject matter relates to a method for producing a modified cell, the method including introducing into the cell the nucleic acid, the vector, the lipid nanoparticle, or the composition disclosed herein. In certain non-limiting embodiments, the presently disclosed subject matter relates to a cell produced by said method.

[0032] In certain non-limiting embodiments, the presently disclosed subject matter relates to a methods of reducing tumor burden in a subject, preventing and / or treating a neoplasm or a tumor in the subject, preventing and / or treating a pathogen infection in a subject, preventing and / or treating an autoimmune disease in a subject, and / or preventing and / or treating an infectious disease in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of the cells or the composition disclosed herein. In certain embodiments, the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject. In certain embodiments, the neoplasm or tumor is cancer. In certain embodiments, the neoplasm or tumor comprises antigen heterogeneity of the antigen. In certain embodiments, the antigen has a low antigen density. In certain embodiments, the antigen is expressed on tumor cells having a low tumor cell frequency.

[0033] In certain embodiments, the neoplasm or tumor is a solid tumor. In certain embodiments, the solid tumor is selected from the group consisting of melanoma, renal cell carcinoma, nonsmall-cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung neuroendocrine carcinoma, small-cell lung cancer, pancreatic cancer, breast cancer, astrocytoma, glioblastoma, laryngeal / pharyngeal carcinoma, EBV-associated nasopharyngeal carcinoma, and ovarian carcinoma.

[0034] In certain embodiments, the neoplasm or tumor is a blood cancer. In certain embodiments, the neoplasm or tumor is a myeloid disorder. In certain embodiments, the myeloid disorder is selected from the group consisting of myelodysplastic syndromes, myeloproliferative neoplasms, chronic myelomonocytic leukemia, or acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelocytic leukemia, and polycythemia vera. In certain embodiments, the myeloid disorder is acute myeloid leukemia (AML).

[0035] In certain embodiments, the neoplasm or tumor is a B-cell malignancy. In certain embodiments, the B-cell malignancy is selected from the group consisting of B cell non-Hodgkin lymphoma (NHL), B cell Hodgkin's lymphoma, B cell acute lymphocytic leukemia (ALL), B cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter’s transformation, and CNS lymphoma. In certain embodiments, the B-cell malignancy is B cell acute lymphocytic leukemia.

[0036] In certain embodiments, the neoplasm or tumor is a leukemia. In certain embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed-phenotype acute leukemia (MLL), hairy cell leukemia, B cell prolymphocytic leukemia, B-cell precursor acute lymphoblastic leukemia, and T-cell precursor acute lymphoblastic leukemia.

[0037] In certain embodiments, the neoplasm or tumor is a lymphoma. In certain embodiments, the lymphoma is selected from the group consisting of Hodgkin’s lymphoma, non-Hodgkin’ s lymphoma, B-cell non-Hodgkin’ s lymphoma, T-cell non-Hodgkin’ s lymphoma, and T-cell precursor acute lymphoblastic lymphoma. In certain embodiments, the subject has a relapse of the neoplasm or tumor. In certain embodiments, the subject received treatment which leads to residual tumor cells.

[0038] In certain non-limiting embodiments, the presently disclosed subject matter relates to the cells or the composition disclosed herein for use in reducing tumor burden, treating and / or preventing a neoplasm or a tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease in a subject. In certain non-limiting embodiments, the presently disclosed subject matter relates to the use of the cells or composition disclosed herein for the manufacturing of a medicament for reducing tumor burden, treating and / or preventing a neoplasm or a tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease in a subject.

[0039] In certain non-limiting embodiments, the presently disclosed subject matter relates to a kit comprising the cells or the composition disclosed herein. In certain embodiments, the kit further comprises written instructions for reducing tumor burden, treating and / or preventing a neoplasm or a tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease.

[0040] BRIEF DESCRIPTION OF THE FIGURES

[0041] 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.

[0042] Figure 1 illustrates different constructs including the pTCR polypeptide.

[0043] Figure 2 illustrates the killing activity of the different constructs in vitro.

[0044] Figures 3A-3C illustrate the activity of the 2A and 4A constructs. Figure 3A shows timecourse FACS analysis of the CAR expression of 2A and 4A constructs upon NALM6 stimulation. Figure 3B shows quantification of bioluminescence imaging of mice receiving cells expressing the 2A and 4A constructs. Figure 3C shows survival curves of mice receiving cells expressing the 2A and 4A constructs.

[0045] Figures 4A-4I illustrate pTCRa incorporation (1A) in 1928z improves T cell expansion and function. Figure 4A shows the features of the constructs designated as 1928z and 1A. Figure 4B shows the CAR surface expression levels of 1928z and 1A. Figure 4C shows FACS analysis of cell division of 1928z or 1A upon NALM6 stimulation. Figure 4D shows a schematic of experimental settings used for determining T cell activities and functionalities by repetitive antigen stimulations. Figure 4E shows the glycolysis and mitochondrial functionality of cells expressing 1928z or 1A. Figure 4F shows the cytokine expression profile of cells expressing 1928z or 1A. Figure 4G shows T cell proliferation upon antigen stimulation of cells expressing 1928z or 1 A obtained from three different donors. Figure 4H shows tumor burden, tumor growth, and survival curves of mice receiving CAR T cells expressing 1928z or 1 A from donor F. Figure 41 shows tumor burden, tumor growth, and survival curves of mice receiving CAR T cells expressing 1928z or 1A from donor R. In NALM6 model, 2e5 19-28z (n=34) and 19-28z-lA (n=12) CAR T cells (Donor R) were infused. The bioluminescent flux indicated tumor volume. Four 19-28z mice and ten 19-28z-lA mice achieved tumor-free and were rechallenged with NALM6 at day 158. Animal survivals were monitored until day 200. The p value was determined by Log-rank (Mantel-Cox) test.

[0046] Figures 5A-5F illustrate 1 A CAR had sustained CAR signaling and activation. Figure 5A shows schematic of the experimental approach used for phospho-proteomics analysis. Figure 5B shows a PCA analysis of the phosphorylation profile of cells expressing 1928z and 1A. Figure 5C shows quantification of the phosphorylation of YBX1 in cells expressing 1928z and 1A. Figures 5D and 5E show immunoblotting of phosphorylated YBX1.

[0047] Figures 6A-6E illustrate the effects of 1 A CAR compared to 1XX CAR. Figure 6A shows expression level of CD 19 in different NALM6 models. Figure 6B shows tumor growth in mice receiving T cells expressing 1928z, 1A, or 1XX at 2e5 dose. Figure 6C shows survival curves of mice receiving T cells expressing 1928z, 1 A, or 1XX at 2e5 dose. Figure 6D shows tumor growth in mice receiving T cells expressing 1A and 1XX at le5 dose. Figure 6E shows survival curves of mice receiving T cells expressing 1A and 1XX at le5 dose.

[0048] Figures 7A and 7B illustrate 1XX-1 A CAR outperforming 1XX CAR. Figure 7A shows immunoblotting of phosphorylated YBX1. Figure 7B shows cumulative fold expansion of T cells expressing 1XX or 1XX-1A CAR.

[0049] Figures 8A-8C illustrate effects of pTCR polypeptide on CCR activity. Figure 8A shows that cells expressing only 1A-CCR had no killing activity. Figures 8B and 8C show the quantification of bioluminescence imaging of different donors receiving cells expressing the HIT70 receptor and the 1 A-CCR.

[0050] Figures 9A and 9B illustrate schematics of representative constructs disclosed herein. Figure 9 A shows the 1 A construct. Figure 9B shows the 1XX-1 A construct.

[0051] Figures 10A-10H illustrate PTCRA (1A) incorporation in 19-28z increases CAR T cell expansion and cytokine secretion. Figure 10A shows a schematic diagram of 19-28z and 19-28z-1A CAR structure. For 19-28z-lA CAR, the truncated PTCRA intracellular domain (1 A domain, 66 aa) was inserted between CD28 and CD3(^. In Figure 10B, four days after NALM6 (5e5) were inoculated in NSG mice, 8e5 TRAC KO 19-28z and 19-28z-lA CAR T cells were infused. The bioluminescent flux represents tumor volume. Figure 10C shows Kaplan Meier curves of NT, 19- 28z, and 19-28z-lA treated mice. The p-value was determined by Log-rank (Mantel-Cox) test. Figure 10D shows the schematic diagram of in vitro repetitive stimulation assay. Every 5 days, CAR T cells were counted, flowed, and co-cultured with NALM6 at a E: T ratio of 3:1. CAR T cells were harvested at day 5, day 10, and day 15 for expansion and functional assays. Figure 10E shows the cumulative fold expansion of 19-28z and 19-28z-lA CAR T cells after repetitive stimulation every five days. Black arrows indicate NALM6 stimulations at a E: T 3:1 ratio. Data were repeated in at least three donors. The p-value was determined by Parametric T-test. Figure 10F shows cytokine productions (IFNg, TNF and IL-2) after 24-hour NALM6 stimulation. Data are represented as mean ± SD. The p-values were determined by Parametric T-test. Figure 10G shows CAR T cells, after three rounds of NALM6 stimulation, subjected to NALM6 stimulation again for 24 hours. The cytokine production of IFNg, TNF, and IL-2 is shown. Data are represented as mean ± SD. The p-values were determined by parametric T-test. Figure 10H shows the cytolytic assays of 19-28z and 19-28z-lA at day 0, day 10 and day 15. CAR T cells and NALM6 co-cultured at indicated E: T ratios for 18 hours. The tumor growth was measured by luciferase assays. For day 15 data, the p-values for 19-28z-l A and 19-28z at 1:4, 1:8 and 1: 16 E: T ratios are 0.0026, 0.0022, and <0.0001 as determined by parametric T-tests.

[0052] Figures 11A-11M depict 19-28z-lA CAR T cells robustly expand in vivo and show enhanced mRNA translation. Figure 11 A shows NALM6 (5e5) injected into NSG mice at day 0. Four days later, 2e5 19-28z, 19-1XX and 19-28z-l A CAR T cells were infused. Bone marrow and spleen samples were harvested after 1-week (day 11), 2-week (day 18), and 6-week (day 45) post CAR T infusion to evaluate CAR T cell persistence. Figure 1 IB shows the tumor cell count, CD8 CAR T cell count, and CD4 CAR T cell count in bone marrow at week 1. Data are represented as mean ± SEM. The p-values were determined by the Mann-Whitney U test. Figure 11C shows CAR T cell count in the spleen at week 1. Data are represented as mean ± SEM. The p-value was determined by the Mann-Whitney U test. Figure 1 ID shows the tumor cell count, CD8 CAR T cell count, and CD4 CAR T cell count in bone marrow at week 2. Data are represented as mean ± SEM. The p-values were determined by the Mann-Whitney U test. Figure 1 IE shows the CAR T cell count in the spleen at week 2. Data are represented as mean ± SEM. The p-value was determined by the Mann-Whitney U test. Figure 1 IF shows the tumor cell count, CD8 CAR T cell count, and CD4 CAR T cell count in bone marrow at week 6. The p-values were determined by the Mann-Whitney U test. Figure 11G shows the CAR T cell count in the spleen at week 6. The p-value was determined by the Mann-Whitney U test. Figure 11H shows the principal component analysis (PCA) of 19-28z, 19-28z-lA, and 19-1XX at the week 1-time point. Figure 111 depicts the heatmap of 19-28z, 19-28z-lA, and 19-1XX at week 1, showing the top 500 differentially expressed genes between 19-1XX and 19-28z-l A. Figure 11 J depicts the PCA plot of 19-28z, 19-28z-lA, and 19-1XX at week 2. Due to the low cell numbers of 19-28z CAR T cells obtained from mice, they were pooled together for sequencing. Figure 1 IK depicts the heatmap of 19-28z, 19-28z-l A, and 19-1XX at week 2, showing the top 500 differentially expressed genes between 19-1XX and 19-28z-lA. Figure 11L depicts the scaled gene expression levels of translation-related genes in 19-28z, 19-28z-lA, and 19-1XX at week 1. Figure 11M depicts the scaled gene expression levels of translation-related genes in 19-28z, 19-28z-lA, and 19-1XX at week 2.

[0053] Figures 12A-12H show 19-28z-lA CAR T cells sustain CAR activation and YBX1 phosphorylation. Figure 12A depicts CAR T cell activation markers CD25, CD69, and 4-1BB after 24-hour NALM6 stimulation. Data are represented as mean ± SD. The p-values were determined by Parametric T test. Data were repeated in three donors. Figure 12B depicts CAR T cell activation markers CD25, CD69, and 4-1BB after 96-hour NALM6 stimulation. Data are represented as mean ± SD. The p-values were determined by Parametric T test. Data were repeated in three donors. Figure 12C depicts a schematic diagram of the 19-28z and 19-28z-lA phosphoproteomics workflow. CAR T cells were stimulated with / without SILAC-labelled NALM6 at an E: T 3:1 ratio. Cells were harvested at 12h and 48h after stimulation. Cell lysates were subjected to phospho-proteomics mass spectrometry. Figure 12D depicts the PCA plot of 19-28z_no_stim, 19-28z-lA_no_stim, 19-28z_12h_stim, 19-28z-lA_12h_stim, 19-28z_48h_stim, and 19-28z-lA_48h_stim samples. Figure 12E depicts the violin plot of 19-28z-lA stim versus 19-28z stim at 12h (green) and 48h (purple) time points. The p-YBXl was highlighted. Figure 12F depicts the pYBXl peptide (SVGDGETVEFDVVEGEK) abundances of 19-28z_no_stim (NS), 19-28z_12h_stim, 19-28z_48h_stim, 19-28z-lA_no_stim (NS), 19-28z-lA_12h_stim, and 19-28z-1 A_48h_stim obtained from the phospho-proteomics mass spectrometry. Data are represented as mean ± SD. Figure 12G depicts western blotting of p-YBXl (Ser 102), YBX1, and GAPDH in 19-28z and 19-28z-lA CAR T cells without or after 12h, 24h, and 48h NALM6 stimulations. Figure 12H depicts western blotting of p-YBXl (Ser 102), YBX1, and GAPDH in 19z and 19-28z CAR T cells without or after 12h, 24h, and 48h NALM6 stimulations.

[0054] Figure 13A-13G shows YBX1 ablation dampens mRNA translation, impairs cell expansion and accelerates exhaustion. Figure 13 A depicts a schematic diagram of NALM6 repetitive stimulation and scRNA-Seq workflow for CTRL-KO and YBX1-KO CAR T cells. Figure 13B shows the cumulative fold expansion of 19-28z CTRL-KO, 19-28z YBX1-KO, 19-28z-l A CTRL-KO, 19-28z-lA YBX1-KO, and 19-28z-lA YBX1-KO + YBX1 CAR T cells after two rounds of NALM6 stimulation every six days. The p-values were determined by the parametric T-test for the last time point. Data were repeated in three donors. Figure 13C depicts the unsupervised Uniform Manifold Approximation and Projection (UMAP) plot of 15 clusters across all samples. Figure 13D shows the individual UMAP of 19-28z CTRL-KO, 19-28z YBX1-KO, 19-28z-l A CTRL-KO, 19-28z-lA YBX1-K0, and 19-28z-lA YBX1-K0 + YBX1 samples. Figure 13E depicts the proportions of 19-28z CTRL-KO, 19-28z YBX1-K0, 19-28z-lA CTRL-KO, 19-28z-lA YBX1-K0, and 19-28z-lA YBX1-K0 + YBX1 within cluster 0 (CD8cm CD8naive-like), cluster 2 (CD4naive-like), cluster 1 (CD8Tex CD8Tem) and cluster 3 (CD8Tex CD8Tpex). Figure 13F shows the top three GSEA enrichment analysis of 19-28z YBX1-K0 versus 19-28z CTRL-KO. Figure 13G shows the top three GSEA enrichment analysis of 19-28z-1 A YBX1-K0 versus 19-28z-l A CTRL-KO.

[0055] Figures 14A-14D depict 1A domain function requires the first two PKC binding motifs. Figure 14A shows the schematic diagram of predicted PKC binding motifs in the 1A domain. Figure 14B shows CAR T cell counts after NALM6 stimulations of 19-28z, 19-28z-lA, 19-28z-1A PBM1-2, and 19-28z-lA PBM1. Data are represented as mean ± SD. The p-values were determined by one-way ANOVA, which compares each column's mean rank with the 19-28z column's mean rank. Figure 14C shows Kaplan Meier curves of NT, 19-28z, 19-28z-l A, 19-28z-1A PBM1-2, and 19-28z-lA PBM1 treated mice in NALM6 mouse model. The p-value was determined by Log-rank (Mantel-Cox) test. Figure 14D shows four days after NALM6 (5e5) inoculated in NSG mice, le5 19-28z, 19-28z-lA, 19-28z-lA PBM1-2, and 19-28z-lA PBM1 CAR T cells were infused. The bioluminescent flux represents tumor volume.

[0056] Figures 15A-15I show 70-28z-lA outperforms 70-28z in RCC and GBM mouse models. Figure 15A depicts the schematic diagram of the orthoptic renal cell carcinoma mouse model. The RCC PDX K5 was surgically implanted in the kidney capsule on day 0 and day 1. On day 7, 2e6 70-28z and 70-28z-l A CAR T cells were infused. Peripheral blood was collected after five weeks post CAR T infusion. The tumor volume was monitored weekly by live imaging. Figure 15B shows peripheral blood hCD45 cell counts of 70-28z and 70-28z-l A mice. Data are represented as mean ± SD. The p-value was determined by Mann-Whitney U test. Figure 15C shows the LNGFR expression levels in hCD45 cells of 70-28z and 70-28z-l A mice. Data are represented as mean ± SD. The p-value was determined by Mann-Whitney U test. Figure 15D shows the percentage of CD4 CAR T cells in 70-28z and 70-28z-lA mice. Data are represented as mean ± SD. The p-value was determined by Mann-Whitney U test. Figure 15E shows the percentage of CD8 CAR T cells in 70-28z and 70-28z-l A mice. Data are represented as mean ± SD. The p-value was determined by Mann-Whitney U test. Figure 15F shows RCC tumor volume of 70-28z and 70-28z-lA mice over time as indicated by the bioluminescent flux. Figure 15F shows Kaplan Meier curves of NT, 70-28z, 70-28z-l A treated mice in RCC model. The p-value was determined by Log-rank (Mantel-Cox) test. Figure 15H shows, at day 0, the GBM cell line U251 (5e5) was transplanted to the right flank of NSG mice. Seven days later, GBM mice received 1.5e5 70-28z and 70-28z-lA CAR T cells. The bioluminescent flux indicates the GBM tumor volumes over time. Figure 151 shows Kaplan Meier curves of NT, 70-28z, 70-28z-lA treated mice in GBM mouse model. The p-value was determined by Log-rank (Mantel-Cox) test.

[0057] Figures 16A-16I show 1A domain further enhances 1XX CAR T cell function. Figure 16A shows western blotting of p-YBXl (Ser 102), YBX1 and GAPDH in 19-1XX and 19-1XX-1 A CAR T cells without or after 12h, 24h, and 48h NALM6 stimulations. Figure 16B shows the cumulative fold expansion of 19-1XX and 19-1XX-1A CAR T cells after multiple rounds of NALM6 stimulation for every six days. Black arrows indicate NALM6 stimulations at a E: T 3:1 ratio. Data were repeated in three donors. The p-value was determined by Parametric T-test for the last time point. Figure 16C shows that, four days after NALM6 (5e5) inoculated in NSG mice, 5e4 19-1XX and 19-1XX-1A CAR T cells were infused. The bioluminescent flux represents tumor volume. Figure 16D shows Kaplan Meier curves of 19-1XX and 19-1XX-1 A treated mice. The p value was determined by Log-rank (Mantel-Cox) test. Figure 16E shows that, at day 0, the GBM cell line U251 (5e5) was transplanted to the right flank of NSG mice. Seven days later, GBM mice received le5 70-1XX and 70-1XX-1 A CAR T cells. The bioluminescent flux indicates the GBM tumor volumes over time. Figure 16F shows the total flux representing tumor burdens at day 27. Data are represented as mean ± SD. The p-value was determined by Mann-Whitney U test. Figure 16G shows quantification on tumor (GFP+) and CAR T cell (hCD45+LNGFR+) percentages of 70-1XX and 70-1XX-1 A mice. Data are represented as mean ± SD. The p-values were determined by the Mann-Whitney U test. Figure 16H shows dot plots for PD1+LAG3+ CAR T cells of 70-1XX and 70-1XX-1 A mice. Figure 161 shows quantification on PD1+LAG3+TIM3+ percentages within 70-1 XX and 70-1XX-1A CAR T cells. Data are represented as mean ± SD. The p-values were determined by the Mann-Whitney U test.

[0058] Figures 17A-17C show in vivo effects in GBM orthotopic models. Figure 17A shows bioluminescence flux of tumors after infusion with the presently disclosed cells. At day 0, U251 cells were stereotactically implanted 2 mm lateral to bregma and 3 mm deep in the brains of NSG mice. Six days later, 5e470-28z (n=4) 70-28z-lA (n=5) CAR T cells were intravenously infused to tumor-bearing mice. Blue, NT; Black, 70-28z; Red, 70-28z-l A. The bioluminescent total flux of 5e4 70-28z and 70-28z-lA at day 35. The p value was determined by Mann-Whitney U test. Figure 17B shows le5 70-28z (n=4) 70-28z-l A (n=5) CAR T cells were intravenously infused to tumor-bearing mice. Figure 17C shows Kaplan Meier curves of NT (n=3), 70-28z 5e4 (n=4), 70-28z-1A 5e4 (n=5), 70-28z le5 (n=4), 70-28z-lA le5 (n=5) treated mice in U251 GBM OR model. The p values were determined by Log-rank (Mantel-Cox) test.

[0059] Figures 18A and 18B show in vivo effects in NALM6 models. Ie5 19-28z (n=9), 19-28z-1A (n=10), 19-1XX (n=10), and 19-1XX-1A (n=10) CART cells were infused. Figure 18A shows the bioluminescent flux in the NALM6 tumor growth over time. Figure 18B shows the bioluminescent total flux of CAR T-treated mice at day 32, 38 and 46. The p values were determined by Kruskal-Wallis test.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] The presently disclosed subject matter provides compositions, e.g., modified immune cells, useful for immunotherapy (e.g., T cell immunotherapy). The presently disclosed subject matter is based, at least in part, on the discovery that antigen-recognizing receptors, including a pre-T cell receptor polypeptide (e.g., pTCR polypeptide), can enhance at least one activity of the cells, e.g., cytotoxicity, cell proliferation, and / or cell persistence.

[0062] Non-limiting embodiments of the presently disclosed subject matter are described by the present specification and Examples.

[0063] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

[0064] 1. Definitions;

[0065] 2. Antigen-Recognizing Receptors;

[0066] 3. Nucleic Acids and Vectors;

[0067] 4. Cells;

[0068] 5. Formulations and Administration;

[0069] 6. Methods of Treatment;

[0070] 7. Kits; and

[0071] 8. Exemplary Embodiments.

[0072] 1. Definitions

[0073] 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 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).

[0074] 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, / .<., 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 20%, e.g., up to 10%, up to 5%, or up to 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 5-fold or within 2-fold, of a value.

[0075] As used herein, a “co-stimulatory molecule” refer to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of lymphocytes to an antigen. In certain embodiments, a co-stimulatory molecule can provide optimal lymphocyte activation.

[0076] As used herein, a “co-stimulatory ligand” refers to a molecule that upon binding to its receptor (e.g., a co-stimulatory molecule) produces a co-stimulatory response, e.g., an intracellular response that effects the stimulation provided when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen.

[0077] By “immunoresponsive cell” is meant a cell that functions in an immune response or a progenitor, or progeny thereof. In certain embodiments, the immunoresponsive cell is a cell of lymphoid lineage. Non-limiting examples of cells of lymphoid lineage include T cells, Natural Killer (NK) cells, B cells, and stem cells from which lymphoid cells may be differentiated. In certain embodiments, the immunoresponsive cell is a cell of myeloid lineage.

[0078] By “activates an immunoresponsive cell” is meant induction of signal transduction 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 tyrosinebased 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 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 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.

[0079] By “stimulates an immunoresponsive cell” is meant a signal that results in a robust and sustained immune response. In various embodiments, this occurs after immune cell (e.g., T-cell) activation or concomitantly mediated through receptors including, but not limited to, CD28, CD137 (4-1BB), 0X40, CD40, ICOS, DAP-10, CD27, NKG2D, CD2, CD150, CD226. 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 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.

[0080] As used herein, the term “antigen heterogeneity” refers to the differential expression of a number of antigens (e.g., tumor antigens, e.g., CD70, CD312) which results in variation in the tumor cell phenotype and distribution of tumor antigen-positive cells.

[0081] As used herein, the term “low antigen density” refers to a target molecule (e.g., an antigen) having a cell surface density of less than about 5,000 molecules per cell. In certain embodiments, the low antigen density is a cell surface density is 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, the low antigen density is a cell surface density is less than about 2,000 molecules per cell. In certain embodiments, the low antigen density is a cell surface density is less than about 1,500 molecules per cell. In certain embodiments, the low antigen density is a cell surface density is less than about 1,000 molecules per cell. In certain embodiments, the low antigen density is a cell surface density is 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 per cell and about 100 molecules per cell.

[0082] As used herein, the term “low tumor cell frequency” refers to a target cell having a target cell frequency of less than about 50% per tumor. In certain embodiments, the low tumor cell frequency is less than about 40% per tumor, less than about 30% per tumor, less than about 20% per tumor, less than about 15% per tumor, less than about 10% per tumor, less than about 5% per tumor, less than about 2% per tumor, or less than about 1% per tumor. In certain embodiments, the low tumor cell frequency is less than about 2% per tumor. In certain embodiments, the low tumor cell frequency is less than about 1.5% per tumor. In certain embodiments, the low tumor cell frequency is less than about 1% per tumor. In certain embodiments, the low tumor cell frequency is between about 40% per tumor and about 20% per tumor, between about 20% per tumor and about 10% per tumor, between about 15% per tumor and about 10% per tumor, between about 20% per tumor and about 5% per tumor, between about 10% per tumor and about 2% per tumor, or between about 10% per tumor and about 1% per tumor.

[0083] The term “antigen-recognizing receptor” as used herein refers to a receptor that is capable of activating and / or stimulating an immune or immunoresponsive cell (e.g., a T-cell) in response to its binding to an antigen. 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 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 Fe 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 native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab’, single chain variable fragment (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 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 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 VL is 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. 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 (Clq) of the classical complement system.

[0084] As used herein, “CDRs” are defined as the complementarity determining region 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 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., etal. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, the CDRs regions are delineated using the PylgClassify system (Adolf-Bryfogle et al., Nucleic acids research 43. DI (2015): D432-D438). As used herein, the term “Linker” shall mean a functional group (e.g., chemical or polypeptide) that covalently attaches two or more polypeptides or nucleic acids so that 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 is a G4S linker. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, which is provided below:

[0085] GGGGSGGGGSGGGGS [ SEQ ID NO: 1 ]

[0086] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, which is provided below:

[0087] GGGGSGGGGSGGGSGGGGS [ SEQ ID NO: 2 ]

[0088] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3, which is provided below:

[0089] GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 3 ]

[0090] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, which is provided below:

[0091] GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [ SEQ ID NO: 4 ]

[0092] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, which is provided below:

[0093] GGGGS [ SEQ ID NO: 5 ]

[0094] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, which is provided below:

[0095] GGGGSGGGGS [SEQ ID NO: 6]

[0096] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion 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 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 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 Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 19973(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 2003 1638(3):257-66).

[0097] 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” 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).

[0098] The term “chimeric antigen receptor” or “CAR” as used herein refers to a molecule comprising an extracellular antigen-binding domain that is fused to an intracellular signaling domain that is capable of activating or stimulating an immune or immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises an scFv. The scFv can be derived from fusing the variable heavy and light regions of an antibody. Alternatively or 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 is selected to have high binding affinity or avidity for the antigen.

[0099] As used herein, the term “substantially identical” or “substantially homologous” refers to a polypeptide or a nucleic acid molecule exhibiting at least about 50% identical or homologous to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleic acid sequence (for example, any 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 about 100% identical or homologous to the amino acid sequence or the nucleic acid sequence used for comparison.

[0100] 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. 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, 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.

[0101] 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)) 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 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 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) 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.

[0102] 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 antigen recognizing receptors (e.g., the extracellular antigen-binding domain of the CAR) comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions and deletions. Modifications can be introduced into the extracellular antigen-binding domain of the presently disclosed CAR 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 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, 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, 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 ( / .<., 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 within a specified sequence or a CDR region are altered.

[0103] 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., neoplasm, and pathogen infection of cell.

[0104] By “effective amount” is meant an amount sufficient to have a therapeutic effect. 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 neoplasm.

[0105] By “endogenous” is meant a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0106] By “exogenous” is meant a nucleic acid molecule or polypeptide that is not endogenously present in a cell. The term “exogenous” would therefore 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 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 non-native promoter or secretory sequence.

[0107] 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.

[0108] By “reduce” is meant to alter negatively by at least about 5%. An alteration may be by about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even by about 100%. The terms “isolated,” “purified,” or “biologically pure” refer to material that is 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 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-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.

[0109] By “isolated cell” is meant a cell that is separated from the molecular and / or cellular components that naturally accompany the cell.

[0110] 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.

[0111] By “neoplasm” or “malignancy” 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. Neoplasm growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasm can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from 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 vagina, or a tissue or cell type thereof. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of the plasma cells). In certain embodiments, the neoplasm is cancer.

[0112] By “specifically binds” is meant a polypeptide or a fragment thereof that recognizes 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- 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 or capable of suppressing an immune response via receptor-ligand binding.

[0113] 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.

[0114] 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 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 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.

[0115] An “individual” or “subject” herein is a vertebrate, such as a human or non-human 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 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.

[0116] As used herein, “a functional fragment” of a molecule or polypeptide includes a fragment of the molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0117] 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.

[0118] 2. Antigen-Recognizing Receptors The presently disclosed subject matter provides antigen-recognizing receptor comprising a pre-T cell receptor (pTCR) polypeptide. In certain embodiments, the antigen-recognizing receptor targets an antigen.

[0119] 2.1. Antigen

[0120] In certain embodiments, the antigen can be a tumor antigen or a pathogen antigen. In certain embodiments, the antigen is a tumor antigen. In certain embodiments, the tumor antigen is an antigen with low antigen density. In certain embodiments, the tumor antigen is expressed on a cell with low tumor cell frequency.

[0121] 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 expressed as a peptide or as an intact protein or a portion thereof. The intact protein or portion thereof can be native or mutagenized. Non-limiting examples of tumor antigens include CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), AD0RA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV) infected cell (e.g. a cell surface antigen), AN09, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135 (FLT3), CD138, CD20, CD22, CD244 (2B4), CD25, CD26, CD276, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNH42, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, EL0VL6, EMB, EMC 10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-63, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, H00K1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, K-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MARTI, GP100, MBOAT1, MBOAT7, melanoma antigen family A, Mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, Mucin 1 (MUC1), Mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D ligands, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), Proteinase3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, R0R1, RYR2, SON, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, ST0N2, SUN3, Survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), Tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

[0122] In certain embodiments, the antigen is selected from the group consisting of CD312, CLEC12A, CD33, CD123, IL1RAP, SIGLEC-6, GRP78, TIM3, CD70, CD20, CD22, CD19, GPRC5D, SLAMF7, BCMA, CD276, and CAIX. In certain embodiments, the antigen is CD 19. In certain embodiments, the antigen is CD70.

[0123] In certain embodiments, the antigen is a pathogen antigen. 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-III / LAV, 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); Togctviridae (e.g. 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 viruses, bunga viruses, phleboviruses and Naira viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnctviridcte 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), 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 =internally transmitted; class 2 =parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses). Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to, Helicobacter pyloris, Borelia burgdorferi, Legionella, Legionella pneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, M. leprae), Staphylococcus aureus, Staphylococcus epidermidis, Neisseria 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., Campylobacter jejuni, Enterococcus sp., Haemophilus influenzae, Bacillus antracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium spp., Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelii. Mycoplasma, Pseudomonas aeruginosa, Pseudomonas fluorescens, Corynobacteria diphtheriae, Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes, Mycoplasma spp., Vibrio cholerae, Borrelia, Francisella, Brucella melitensis, Proteus mirabilis, and Proteus.

[0124] 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.

[0125] 2.2. Chimeric Antigen Receptors

[0126] In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR). CARs are engineered receptors, which graft or confer a specificity of interest onto an immune effector cell. CARs can be used to graft the specificity of a monoclonal antibody onto a T cell; with transfer of their coding sequence facilitated by retroviral vectors.

[0127] There are three generations of CARs. “First generation” CARs are typically composed of an extracellular antigen-binding domain (e.g., an scFv) that binds to a target antigen, and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+and CD8+T cells through their CD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. “Second generation” CARs include a signaling domain of a co-stimulatory molecule (e.g., CD28, 4-1BB, ICOS, 0X40, CD27, CD40, NKG2D, DAP- 10, CD2, CD 150, CD226) to the intracellular signaling domain of the CAR to provide co-stimulation signals to the cell (e.g., T cell or NK cell). “Second generation” CARs comprise those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3Q. “Third generation” CARs comprise those that provide multiple co-stimulation (e.g., CD28 and 4-1BB) and activation (CD3Q.

[0128] In certain embodiments, the antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to the first antigen, and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. In certain embodiments, the CAR further comprises a hinger / spacer region.

[0129] 2.2.1. Extracellular Antigen-Binding Domain

[0130] In certain embodiments, the extracellular antigen-binding domain of the CAR (for example, an scFv) binds to the antigen with a dissociation constant (KD) of about 5 x 10'7M or less, about 1 x 10'7M or less, about 5 x 10'8M or less, about 1 x 10'8M or less, about 5 x 10'9M or less, or about 1 x 10'9M or less, or about 1 x 10'10M or less. In certain embodiments, the extracellular antigen-binding domain of the CAR (for example, an scFv) binds to the antigen with a KD of about 1 x 10'8M or less.

[0131] Binding of the extracellular antigen-binding domain (for example, in an scFv) 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-antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody, or an scFv) specific for the complex of interest. For example, the scFv 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 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 antigenbinding domain of the CAR 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 fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0132] The extracellular antigen-binding domain can comprise or be an scFv, a Fab (which is optionally crosslinked), or a F(ab)2. In certain embodiments, any of the foregoing molecules may be comprised in a fusion protein with a heterologous sequence to form the extracellular antigenbinding domain. In certain embodiments, the extracellular antigen-binding domain comprises or is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a murine scFv.

[0133] In addition, the extracellular antigen-binding domain of the CAR can comprise a leader or a signal peptide that directs the nascent protein into the endoplasmic reticulum. Signal peptide or leader can be essential if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids long) present at the N-terminus of newly synthesized proteins that directs their entry to the secretory pathway. In certain embodiments, the signal peptide is covalently joined to the 5’ terminus (N-terminus) of the extracellular antigen-binding domain of the CAR. Exemplary leader sequences include, but is not limited to, a human IL-2 signal sequence (e.g., a human IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 7), a mouse IL-2 signal sequence (e.g., a mouse IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 8); a human kappa leader sequence (e.g., a human kappa leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9), a mouse kappa leader sequence (e.g., a mouse kappa leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10); a human CD8 leader sequence (e.g., a human CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 11); a truncated human CD8 signal peptide (e.g., a truncated human CD8 signal peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 12); a human albumin signal sequence (e.g., a human albumin signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 13); and a human prolactin signal sequence (e.g., a human prolactin signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 14). SEQ ID NO: 7-14 are provided below.

[0134] MYRMQLLSCIALSLALVTNS [SEQ ID NO: 7 ]

[0135] MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 8 ]

[0136] METPAQLLFLLLLWLPDTTG [SEQ ID NO: 9]

[0137] METDTLLLWVLLLWVPGSTG [SEQ ID NO: 10 ]

[0138] MALPVTALLLPLALLLHAARP [ SEQ ID NO: 11 ]

[0139] MALPVTALLLPLALLLHA [SEQ ID NO: 12 ]

[0140] MKWVTFISLLFSSAYS [SEQ ID NO: 13 ]

[0141] MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 14 ] In certain embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12.

[0142] 2.2.1.1. Exemplary Extracellular Antigen-Binding Domains

[0143] In certain embodiments, the antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD312.

[0144] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 72 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 74, a conservative modification thereof; a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 72, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 74; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 75, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 76, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 77.

[0145] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1, a CDR2, and a CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 78; and a VL comprising a CDR1, a CDR2, and a CDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 79.

[0146] In certain embodiments, the extracellular antigen-binding domain of the CAR 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 the amino acid sequence set forth in SEQ ID NO: 78, 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 the amino acid sequence set forth in SEQ ID NO: 79. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises VH comprising the amino acid sequence set forth in SEQ ID NO: 78 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 79. In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 80. SEQ ID NOs: 72-80 are provided in the following Table 1. In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0147] Table 1

[0148]

[0149] In certain embodiments, the antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD276.

[0150] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 81 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83, a conservative modification thereof; a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 81, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 84, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 86.

[0151] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1, a CDR2, and a CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 87; and a VL comprising a CDR1, a CDR2, and a CDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 88.

[0152] In certain embodiments, the extracellular antigen-binding domain of the CAR 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 the amino acid sequence set forth in SEQ ID NO: 87, 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 the amino acid sequence set forth in SEQ ID NO: 88. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises VH comprising the amino acid sequence set forth in SEQ ID NO: 87 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0153] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89. SEQ ID NOs: 81-89 are provided in the following Table 2. In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0154] Table 2

[0155]

[0156]

[0157] In certain embodiments, the antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD22.

[0158] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 90 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 91 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 92, a conservative modification thereof; a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 90, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 91, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 92; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95.

[0159] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1, a CDR2, and a CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 96; and a VL comprising a CDR1, a CDR2, and a CDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 97.

[0160] In certain embodiments, the extracellular antigen-binding domain of the CAR 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 the amino acid sequence set forth in SEQ ID NO: 96, 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 the amino acid sequence set forth in SEQ ID NO: 97. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises VH comprising the amino acid sequence set forth in SEQ ID NO: 96 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 97.

[0161] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 98. SEQ ID NOs: 90-98 are provided in the following Table 3. In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0162] Table 3

[0163]

[0164] In certain embodiments, the antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD70.

[0165] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 99 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101, a conservative modification thereof; a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 103 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 99, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 103, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104.

[0166] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1, a CDR2, and a CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 105; and a VL comprising a CDR1, a CDR2, and a CDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 106.

[0167] In certain embodiments, the extracellular antigen-binding domain of the CAR 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 the amino acid sequence set forth in SEQ ID NO: 105, 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 the amino acid sequence set forth in SEQ ID NO: 106. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises VH comprising the amino acid sequence set forth in SEQ ID NO: 105 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 106. SEQ ID NOs: 99-106 are provided in the following Table 4. In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0168] Table 4

[0169]

[0170]

[0171] In certain embodiments, the antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD 19.

[0172] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109, a conservative modification thereof; a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112 or a conservative modification thereof. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 107, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109; and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112.

[0173] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VH comprising a CDR1, a CDR2, and a CDR3 of the VH having the amino acid sequence set forth in SEQ ID NO: 113; and a VL comprising a CDR1, a CDR2, and a CDR3 of the VL having the amino acid sequence set forth in SEQ ID NO: 114.

[0174] In certain embodiments, the extracellular antigen-binding domain of the CAR 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 the amino acid sequence set forth in SEQ ID NO: 113, 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 the amino acid sequence set forth in SEQ ID NO: 114. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises VH comprising the amino acid sequence set forth in SEQ ID NO: 113 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 114.

[0175] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv that comprises or consists of the amino acid sequence set forth in SEQ ID NO: 115. SEQ ID NOs: 107-115 are provided in the following Table 5. In certain embodiments, the VH and VL are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0176] Table 5

[0177]

[0178] The VH and / or VL amino acid sequences having at least about 80%, 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%, or about 99%) homology or identity to a specific sequence (e.g., SEQ ID NOs: 78, 79, 87, 88, 96, 97, 105, 106, 113, and 114) 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., CD312, CD276, CD22, CD70, CD19). 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: 78, 79, 87, 88, 96, 97, 105, 106, 113, and 114). In certain embodiments, 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 of the CAR comprises VH and / or VL sequence selected from SEQ ID NOs: 78, 79, 87, 88, 96, 97, 105, 106, 113, and 114, including post-translational modifications of that sequence (SEQ ID NO: 78, 79, 87, 88, 96, 97, 105, 106, 113, and 114).

[0179] 2.2.2. Transmembrane Domain and Hinge / Spacer Region

[0180] In certain embodiments, the CAR comprises a transmembrane domain. Different transmembrane domains result in different receptor stability. After antigen recognition, receptors cluster and a signal are transmitted to the cell. In accordance with the presently disclosed subject matter, the transmembrane domain of the antigen-recognizing receptor can comprise a native or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3(^ polypeptide, a CD40 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, a CD84 polypeptide, a CD 166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an IC M-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof.

[0181] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises a transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of the amino acid sequence having a NCBI Reference No: NP_006130 (SEQ ID NO: 15), which is at least about 20, or at least about 25, or at least about 30, and / or up to about 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of 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, 153 to 179, or 200 to 220 of SEQ ID NO: 15. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide that comprises or consists of amino acids 153 to 179 of SEQ ID NO: 15. SEQ ID NO: 15 is provided below.

[0182] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYG NYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHL CPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS [SEQ ID NO: 15 ]

[0183] In certain embodiments, the CAR further comprises a hinge / spacer region that links the extracellular antigen-binding domain to the transmembrane 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 embodiments, the hinge / spacer region of the CAR can comprise a native or modified hinge region of a CD8 polypeptide, a CD28 polypeptide, a CD3(^ polypeptide, a CD40 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, a CD84 polypeptide, a CD 166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an IC M-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof. 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 (e.g., a portion of SEQ ID NO: 15), a portion of aCD8 polypeptide, or a synthetic spacer sequence.

[0184] In certain embodiments, the CAR further comprises a hinge / spacer region comprising a native or modified hinge region of a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the first antigen-recognizing receptor (e.g., a CAR) comprises a CD28 polypeptide comprising or consisting of amino acids 114 to 152 of SEQ ID NO: 15.

[0185] In certain embodiments, the hinge / spacer region is positioned between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge / spacer region comprises a CD8 polypeptide, a CD28 polypeptide, a CD3(^ polypeptide, a CD4 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, a CD 166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3(^ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an 0X40 polypeptide, a CD 166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, a NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with the immune response), or a combination thereof.

[0186] In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and the hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD84 polypeptide and the transmembrane domain comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD 166 polypeptide and the transmembrane domain comprises a CD 166 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8a polypeptide and the transmembrane domain comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8b polypeptide and the transmembrane domain comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises an ICOS polypeptide.

[0187] 2..3. Intracellular Signaling Domain

[0188] In certain embodiments, the CAR comprises an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a pre-T cell receptor (pTCR) polypeptide. In certain embodiments, the pTCR polypeptide is a pTCRa polypeptide. In immature T cells, the T cell receptor beta-chain gene (TCRB) is rearranged and expressed before the T cell receptor alpha-chain gene (TCRA). At this early stage, TCRB can associate with the pre-T cell receptor alpha chain (pTCRa). The pTCRa, together with TCRB and the CD3 complex, makes up the pre-T cell receptor (pre-TCR), which regulates T cell development.

[0189] In certain embodiments, the intracellular signaling domain of the CAR comprises a pTCR polypeptide. In certain embodiments, the pTCR polypeptide is a human pTCR. In certain embodiments, the human pTCR polypeptide comprises or consists of the amino acid sequence set forth in UniProt Reference No. Q6ISU1 (SEQ ID NO: 16) or a fragment thereof. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 1 to 281, 24 to 281, 28 to 146, 147 to 281, 147 to 167, 168 to 281, or 168 to 233 of SEQ ID NO: 16. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 281 of SEQ ID NO: 16. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 233 of SEQ ID NO: 16. SEQ ID NO: 16 is provided below:

[0190] MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPI FSAGNGSA LDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQPMHLSGEASTARTCPQEPL RGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSS PRPQPRDRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGA

[0191] A [SEQ ID NO: 16]

[0192] In certain embodiments, the pTCR polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, which is provided below: TCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEG SYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA [SEQ ID NO: 17 ]

[0193] In certain embodiments, the pTCR polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, which is provided below:

[0194] TCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPV

[0195] [SEQ ID NO: 18 ]

[0196] In certain embodiments, the pTCR polypeptide is a mouse pTCR polypeptide. In certain embodiments, the mouse pTCR polypeptide comprises or consists of the amino acid sequence set forth in UniProt Reference No. P0C6B2 (SEQ ID NO: 19) or a fragment thereof. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 199 of SEQ ID NO: 19. SEQ ID NO: 19 is provided below:

[0197] MARTWLLLLLGVRCQALPSGIAGTPFPSLAPPITLLVDGRQHMLVVCLVLDAAPPGLDNPVWFSAGNGSA LDAFTYGPSLAPDGTWTSLAQLSLPSEELEAWEPLVCHTRPGAGGQNRSTHPLQLSGESSTARSCFPEPL GGTQRQVLWLSLLRLLLFKLLLLDVLLTCSHLRLHVLAGQHLQPPPSRKSLPPTHRIWT [SEQ ID NO: 19]

[0198] In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3(^ polypeptide. CD3(^ can activate or stimulate a cell (e.g., a cell of the lymphoid lineage, e.g., a T-cell). Wild type (“native”) CD3(^ comprises three functional immunoreceptor tyrosine-based activation motifs (IT AMs), three functional basic-rich stretch (BRS) regions (BRS1, BRS2 and BRS3). CD3(^ transmits an activation signal to the cell (e.g., a cell of the lymphoid lineage, e.g., a T-cell) after antigen is bound. The intracellular signaling domain of the CD3^-chain is the primary transmitter of signals from endogenous TCRs.

[0199] In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3(^. In certain embodiments, the native CD3(^ comprises or consists of 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% identical or homologous to the amino acid sequence having a NCBI Reference No: NP 932170 (SEQ ID NO: 20) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3(^ polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 20, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, the native CD3(^ comprises or consists of the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 20. In certain embodiments, the intracellular signaling domain of the CAR comprises a native CD3(^ comprising or consisting of the amino acid sequence of amino acids 52 to 164 of SEQ ID NO: 20. SEQ ID NO: 20 is provided below: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 20 ]

[0200] In certain embodiments, the native CD3(^ comprises or consists of 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% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO: 21 is provided below:

[0201] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 21 ]

[0202] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide. In certain embodiments, the modified CD3(^ polypeptide comprises one, two or three ITAMs. In certain embodiments, the modified CD3(^ polypeptide comprises a native IT AMI. In certain embodiments, the native IT AMI comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0203] QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 22 ]

[0204] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 22 is set forth in SEQ ID NO: 23, which is provided below.

[0205] CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA

[0206] [SEQ ID NO: 23]

[0207] In certain embodiments, the modified CD3(^ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss of function mutations comprises a mutation of a tyrosine residue in ITAM1. In certain embodiments, the IT AMI variant consists of two loss-of-function mutations. In certain embodiments, the IT AMI variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24, which is provided below.

[0208] QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 24 ]

[0209] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 is set forth in SEQ ID NO: 25, which is provided below.

[0210] CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA

[0211] [SEQ ID NO: 25 ]

[0212] In certain embodiments, the modified CD3(^ polypeptide comprises a native ITAM2. In certain embodiments, the native IT M2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26, which is provided below. QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 26]

[0213] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 26 is set forth in SEQ ID NO: 27, which is provided below.

[0214] CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA

[0215] [SEQ ID NO: 27 ]

[0216] In certain embodiments, the modified CD3(^ polypeptide comprises an ITAM2 variant. In certain embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) the loss of function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28, which is provided below.

[0217] QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 28 ]

[0218] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 28 is set forth in SEQ ID NO: 29, which is provided below.

[0219] CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAA

[0220] [SEQ ID NO: 29]

[0221] In certain embodiments, the modified CD3(^ polypeptide comprises a native ITAM3. In certain embodiments, the native ITAM3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30, which is provided below.

[0222] HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 30 ]

[0223] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 30 is set forth in SEQ ID NO: 31, which is provided below.

[0224] CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG

[0225] [SEQ ID NO: 31]

[0226] In certain embodiments, the modified CD3(^ polypeptide comprises an ITAM3 variant. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) the loss of function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32, which is provided below.

[0227] HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 32 ] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 32 is set forth in SEQ ID NO: 33, which is provided below.

[0228] CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAG

[0229] [SEQ ID NO: 33]

[0230] Various modified CD3(^ polypeptides and CARs comprising modified CD3(^ polypeptides are disclosed in International Patent Application Publication No. WO2019 / 133969, which is incorporated by reference hereby in its entirety.

[0231] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising a native IT AMI, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 22, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO: 28, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO: 32. In certain embodiments, the CAR is designated as “1XX”. In certain embodiments, the modified CD3(^ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34. SEQ ID NO: 34 is provided below:

[0232] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEA FSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 34 ]

[0233] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3(^ polypeptide comprising or consisting of 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%, or at least about 99%, at least about 100% identical to SEQ ID NO: 34 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions.

[0234] An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 34 is set forth in SEQ ID NO: 35, which is provided below.

[0235] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGC TCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGG AAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCC TTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGGCTCA GTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 35 ]

[0236] In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one co-stimulatory signaling region. In certain embodiments, the at least one costimulatory region comprises a co-stimulatory molecule or a portion thereof. In certain embodiments, the at least one co-stimulatory region comprises at least an intracellular domain of at least one co-stimulatory molecule or a portion thereof. Non-limiting examples of costimulatory molecules include CD28, 4- IBB, 0X40, CD27, CD40, CD 154, CD97, CDlla / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0237] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide, e.g., an intracellular domain of CD28 or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises an intracellular domain of human CD28 or a portion thereof.

[0238] In certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region of the first antigen-recognizing receptor comprise or consists of 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%, or at least about 99%, at least about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 15 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region of the CAR comprises or consist of an amino acid sequence that is a consecutive portion of SEQ ID NO: 15, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region of the CAR comprises or consists of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises a CD28 polypeptide comprising or consisting of amino acids 180 to 220 of SEQ ID NO: 15.

[0239] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180 to 220 of SEQ ID NO: 15 is set forth in SEQ ID NO: 36, which is provided below.

[0240] AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCC GCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 36] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region that comprises an intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region comprises or consists of 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%, or at least about 99%, at least about 100% identical or homologous to the amino acid sequence having a NCBI Reference No: NP 031668.3 (or SEQ ID NO: 37) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 37, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprised in the co-stimulatory signaling region of the CAR comprises or consists of the amino acid sequence of amino acids 1 to 218, 1 to 50, 50 to 100, 100 to 150, 150 to 218, 178 to 218, or 200 to 218 of SEQ ID NO: 37. In certain embodiments, the co-stimulatory signaling region of the CAR comprises a CD28 polypeptide that comprises or consists of amino acids 178 to 218 of SEQ ID NO: 37. SEQ ID NO: 37 is provided below.

[0241] MTLRLLFLALNFFSVQVTENKILVKQSPLLVVDSNEVSLSCRYSYNLLAKEFRASLYKGVNSDVEVCVGN GNFTYQPQFRSNAEFNCDGDFDNETVTFRLWNLHVNHTDIYFCKIEFMYPPPYLDNERSNGTIIHIKEKH LCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPA RDFAAYRP [SEQ ID NO: 37 ]

[0242] In certain embodiments, the intracellular signaling domain of the CAR comprises a co-stimulatory signaling region that comprises a 4-1BB polypeptide, e.g., an intracellular domain of 4- IBB or a portion thereof. In certain embodiments, the co-stimulatory signaling region comprises an intracellular domain of human 4-1BB or a portion thereof. In certain embodiments, the 4-1BB comprised in the co-stimulatory signaling region of the CAR comprises or consists of 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%, or at least about 99%, at least about 100% identical or homologous to the sequence having a NCBI Ref. No.: NP_001552 (SEQ ID NO: 38) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the 4- IBB comprised in the co-stimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 38, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 200, or up to about 255 amino acids in length. In certain embodiments, the co-stimulatory signaling region of the CAR comprises a 4-1BB polypeptide that comprises or consists of the amino acid sequence of amino acids 1 to 255, 1 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 255 of SEQ ID NO: 38. In certain embodiments, the co-stimulatory signaling region of the CAR comprises a 4- IBB polypeptide comprising or consisting of the amino acid sequence of amino acids 214 to 255 of SEQ ID NO: 38. SEQ ID NO: 38 is provided below.

[0243] MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKG VFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCS LDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRF SVVKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 38 ]

[0244] In certain embodiments, the intracellular signaling domain of the CAR comprises two co-stimulatory signaling regions, wherein the first co-stimulatory signaling region comprises an intracellular domain of a first co-stimulatory molecule or a portion thereof, and the second co-stimulatory signaling region comprises an intracellular domain of a second co-stimulatory molecule or a portion thereof. The first and second co-stimulatory molecules are independently selected from the group consisting of CD28, 4- IBB, 0X40, CD27, CD40, CD 154, CD97, CD1 la / CD18, ICOS, DAP- 10, CD2, CD 150, CD226, and NKG2D. In certain embodiments, the intracellular signaling domain of the CAR comprises two co-stimulatory signaling regions, wherein the first co-stimulatory signaling region comprises an intracellular domain of CD28 or a portion thereof and the second co-stimulatory signaling region comprises an intracellular domain of 4- IBB or a portion thereof.

[0245] 2.2.4. Exemplary CARs

[0246] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “28z-lA” or “1A” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 116. SEQ ID NO: 116 is provided below.

[0247] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTC SCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 116]

[0248] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD 19, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “1928z-lA” or “19-1A ” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 117. SEQ ID NO: 117 is provided below.

[0249] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIY PGDGDTNYNGKFKGQATLT ADKS S STAYMQL SGLT SE DS AVY FCARKT I S S VVDFY FDY GQGTT VT VS S GGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQ PRDRRWGDTPPGRKPGSPVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [ SEQ ID NO: 117 ]

[0250] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD70, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “7028z-lA” or “70-1A.” In certain embodiments, the CAR comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 132. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 132. SEQ ID NO: 132 is provided below.

[0251] MELGLSWIFLLAILKGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAPGKGLEWVAVI SYDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVTVSSGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPLTFGGGTKVEIKAAAIEVMYPPPYLDNEKSNGT IIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDR RWGDTPPGRKPGSPVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 132 ] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “28z-3A” or “3 A.” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 118. SEQ ID NO: 118 is provided below.

[0252] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTC SCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEGSY LSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAARVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR [SEQ ID NO: 118 ]

[0253] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD 19, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “1928z-3A” or “19-3 A.” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 119. SEQ ID NO: 119 is provided below.

[0254] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIY PGDGDTNYNGKFKGQATLT ADKS S STAYMQL SGLT SE DS AVY FCARKT I S S VVDFY FDY GQGTT VT VS S GGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQ PRDRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAARVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 119]

[0255] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, (b) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “28z-2A” or “2A” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 120. SEQ ID NO: 120 is provided below.

[0256] FWVLVVVGGVLACYSLLVTVAFIIFWVTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSS PRPQPRDRRWGDTPPGRKPGSPVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 120]

[0257] In certain embodiments, the first antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD 19, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, (b) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “1928z-2A” or “19-2A ” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 121. SEQ ID NO: 121 is provided below.

[0258] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIY PGDGDTNYNGKFKGQATLT ADKS S STAYMQL SGLT SE DS AVY FCARKT I S S VVDFY FDY GQGTT VT VS S GGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVTCSCLCDPAGPLPSPATT TRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [ SEQ ID NO: 121 ] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, (b) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “28z-4A” or “4A.” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 122. SEQ ID NO: 122 is provided below.

[0259] FWVLVVVGGVLACYSLLVTVAFIIFWVTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSS PRPQPRDRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGA ARSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR [SEQ ID NO: 122 ]

[0260] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD 19, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a native CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, (b) a costimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (c) a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the CAR is designated as “1928z-4A” or “19-4A ” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 123. SEQ ID NO: 123 is provided below.

[0261] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIY PGDGDTNYNGKFKGQATLT ADKS S STAYMQL SGLT SE DS AVY FCARKT I S S VVDFY FDY GQGTT VT VS S GGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVTCSCLCDPAGPLPSPATT TRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEGSYLSSYPTCPAQAWCSRS ALRAPSSSLGAFFAGDLPPPLQAGAARSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 123 ]

[0262] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a modified CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (c) a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAR is designated as “28zlXX-lA” or “1XX-1A” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 124. SEQ IDNO: 124 is provided below.

[0263] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTC SCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGM KGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 124 ]

[0264] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD 19, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a modified CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (c) a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAR is designated as “1928zlXX-lA” or “191XX-1A.” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 125. SEQ ID NO: 125 is provided below.

[0265] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIY PGDGDTNYNGKFKGQATLT ADKS S STAYMQL SGLT SE DS AVY FCARKT I S S VVDFY FDY GQGTT VT VS S GGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQ PRDRRWGDTPPGRKPGSPVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 125 ] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD70, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a modified CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, and (c) a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAR is designated as “7028zlXX-lA” or “701XX-1A.” In certain embodiments, the CAR comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 133. In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 133. SEQ ID NO: 133 is provided below.

[0266] MELGLSWIFLLAILKGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYIMHWVRQAPGKGLEWVAVI SYDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDTDGYDFDYWGQGTLVTVSSGGG GSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRTNWPLTFGGGTKVEIKAAAIEVMYPPPYLDNEKSNGT IIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHYQPYAPPRDFAAYRSTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDR RWGDTPPGRKPGSPVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 133 ]

[0267] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a modified CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain comprises a native CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, and (c) a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAR is designated as “28zlXX-3A” or “1XX-3A”

[0268] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a modified CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18, (b) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (c) a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAR is designated as “28zlXX-2A” or “1XX2A.”

[0269] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising (a) a pTCR polypeptide, (b) a modified CD3(^ polypeptide, and (c) a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, the intracellular signaling domain comprises a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the intracellular signaling domain comprises a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 17, (b) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (c) a modified CD3(^ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the CAR is designated as “28zlXX-4A” or “1XX4A.”

[0270] 2.3. Chimeric Ligand Receptors

[0271] In certain embodiments, the antigen-recognizing receptor is a chimeric ligand receptor that comprises a ligand or a portion thereof that binds to the antigen. In certain embodiments, the chimeric ligand receptor further comprises a transmembrane domain and an intracellular signaling domain.

[0272] In certain embodiments, the transmembrane domain is fused to the ligand or portion thereof. In certain embodiments, the transmembrane domain is fused to the intracellular signaling domain. In certain embodiments, the transmembrane domain is positioned between the ligand or portion thereof and the intracellular signaling domain. In certain embodiments the transmembrane domain of the chimeric ligand receptor is a transmembrane domain disclosed in Section 2.2.2. In certain embodiments, the intracellular signaling domain of the chimeric ligand receptor comprises a pTCR polypeptide (e.g., as disclosed in Section 2.2.3).

[0273] Additional information on the presently disclosed chimeric ligand receptor can be found in Sauer et al., Blood (2021) 138 (4): 318-330, the content of which is incorporated by reference in its entirety.

[0274] 2.4. TCR-Like Fusion Molecules

[0275] In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-Independent TCR-based Chimeric Antigen Receptor (also known as “HIT”, e.g., those disclosed in International Patent Application No. PCT / US19 / 017525, which is incorporated by reference in its entirety), and T cell receptor fusion constructs (TRuCs) (e.g., those disclosed in Baeuerle et al., “Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response,” Nature Communications volume 10, Article number: 2087 (2019), which is incorporated by reference in its entirety).

[0276] In certain embodiments, the TCR-like fusion molecule is a recombinant T cell receptor (TCR). In certain embodiments, the recombinant TCR comprises at least one antigen-binding chain. In certain embodiments, the antigen-binding domain of the recombinant TCR comprises 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 recombinant TCR comprises two antigen binding chains, i.e., a first antigen binding chain and a second antigen binding chain. In certain embodiments, the first and second antigen-binding chains each comprises a constant domain. In certain embodiments, the recombinant TCR binds to an antigen (e.g., a first antigen or a second antigen) in an HLA-independent manner. Thus, in certain embodiments, the recombinant TCR is an HLA-independent (or non-HLA restricted) TCR (referred to as “HIT”).

[0277] In certain embodiments, the first antigen-binding chain comprises an antigen-binding fragment of a heavy chain variable region (VH) of an antibody. In certain embodiments, the second antigen-binding chain comprises an antigen-binding fragment of a light chain variable region (VL) of an antibody. In certain embodiments, the first antigen-binding chain comprises an antigenbinding fragment of a VH of an antibody, and the second antigen-binding chain comprises an antigen-binding fragment of a VL of the antibody.

[0278] In certain embodiments, the constant domain comprises a TCR constant region selected from the group consisting of a native or modified TRAC polypeptide, a native or modified TRBC polypeptide, a native or modified TRDC polypeptide, a native or modified TRGC polypeptide and any variants or functional fragments thereof. In certain embodiments, the constant domain comprises a native or modified TRAC polypeptide. In certain embodiments, the constant domain comprises a native or modified TRBC polypeptide. In certain embodiments, the first antigenbinding chain comprises a TRAC polypeptide, and the second antigen-binding chain comprises a TRBC polypeptide. In certain embodiments, the first antigen-binding chain comprises a TRBC polypeptide, and the second antigen-binding chain comprises a TRAC polypeptide.

[0279] In certain embodiments, the first antigen-binding chain comprises a VH of an antibody and a TRAC polypeptide, and the second antigen-binding chain comprises a VL of an antibody and a TRBC polypeptide.

[0280] In certain embodiments, the first antigen-binding chain comprises a VH of an antibody and a TRBC polypeptide, and the second antigen-binding chain comprises a VL of an antibody and a TRAC polypeptide. In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the TRAC polypeptide is endogenous. In certain embodiments, the TRBC polypeptide is endogenous. In certain embodiments, both the TRAC polypeptide and the TRBC polypeptide are endogenous.

[0281] In certain embodiments, the antigen binding chain is capable of associating with a CD3(^ polypeptide. In certain embodiments, the antigen binding chain, upon binding to an antigen, is capable of activating the CD3(^ polypeptide associated to the antigen binding chain. In certain embodiments, the activation of the CD3(^ polypeptide is capable of activating an immunoresponsive cell. In certain embodiments, the TCR-like fusion molecule is capable of integrating with a CD3 complex and providing HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces an endogenous TCR in a CD3 / TCR complex.

[0282] In certain embodiments, the first and second antigen binding chains bind to an antigen with a dissociation constant (KD) of about 2 * 10'7M or less. In certain embodiments, the first and second antigen binding chains bind to an antigen with a high binding affinity. In certain embodiments, the Kois about 2 x 10’7M or less, about 1 x 10’7M or less, about 9 x 10’8M or less, about 1 x 10'8M or less, about 9 x 1 O’9M or less, about 5 x 10'9M or less, about 4 x 10'9M or less, about 3 x 10'9or less, about 2 xiO’9M or less, or about 1 x 10'9M or less. In certain embodiments, the Kois about 1 x 10'8M or less. In certain embodiments, the Kois about 3 x 1 O’9M or less. In certain embodiments, the Kois about 5 x 10'9M or less. In certain embodiments, the Kois from about 1 x 10'9M to about 1 x 10'8M. In certain embodiments, the Kois from about 1.5 x io-9M to about 1 x 10'8M. In certain embodiments, the Kois from about 5 x 10'9M to about 1x10'8M.

[0283] In certain embodiments, the constant domain comprises a TCR constant region, e.g., T cell receptor alpha constant region (TRAC), T cell 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.

[0284] In certain embodiments, the first antigen binding chain or the second antigen binding chain comprises a constant domain that comprises a native or modified TRAC polypeptide. In certain embodiments, the TRAC 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: 39 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39. SEQ ID NO: 39 is provided below.

[0285] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDF ACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0286] [SEQ ID NO: 39]

[0287] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 39 is set forth in SEQ ID NO: 40, which is provided below.

[0288] ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTAT TCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAAC TGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTT GCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCT GTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGAT TGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

[0289] [SEQ ID NO: 40]

[0290] In certain embodiments, the TRAC 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: 41 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 41. SEQ ID NO: 41 is provided below.

[0291] IPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNK SDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRL WSS [SEQ ID NO: 41]

[0292] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 41 is set forth in SEQ ID NO: 42, which is provided below.

[0293] ATTCCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTG TCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCAC AGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAA TCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAG AAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCT GTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTG TGGTCCAGC [SEQ ID NO: 42 ]

[0294] In certain embodiments, the TRAC polypeptide comprises or consists of 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 the gene of NCBI Genbank ID: 28755, NG_001332.3, range 925603 to 930229 (SEQ ID NO: 43) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 43. SEQ ID NO: 43 is provided below.

[0295] ATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCT ATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAA ACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACT TTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGG CAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCA ATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTA AGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAAGGTGGC AGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTG TTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCC TGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCATTAACCCACCAATCAC TGATTGTGCCGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGGGGTGTGC CCAGAGGAAGCACCATTCTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATT GGAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGA AGAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGA GCTCAATGAGAAAGGAGAAGAGCAGCAGGCATGAGTTGAATGAAGGAGGCAGGGCCGGGTCACAGGGCCT TCTAGGCCATGAGAGGGTAGACAGTATTCTAAGGACGCCAGAAAGCTGTTGATCGGCTTCAAGCAGGGGA GGGACACCTAATTTGCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTTGCTCTT GTTGCCCAGGCTGGAGTGCAATGGTGCATCTTGGCTCACTGCAACCTCCGCCTCCCAGGTTCAAGTGATT CTCCTGCCTCAGCCTCCCGAGTAGCTGAGATTACAGGCACCCGCCACCATGCCTGGCTAATTTTTTGTAT TTTTAGTAGAGACAGGGTTTCACTATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCACC CGCTTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCACACCCGGCCTGCTTTTCTTAAAGAT CAATCTGAGTGCTGTACGGAGAGTGGGTTGTAAGCCAAGAGTAGAAGCAGAAAGGGAGCAGTTGCAGCAG AGAGATGATGGAGGCCTGGGCAGGGTGGTGGCAGGGAGGTAACCAACACCATTCAGGTTTCAAAGGTAGA ACCATGCAGGGATGAGAAAGCAAAGAGGGGATCAAGGAAGGCAGCTGGATTTTGGCCTGAGCAGCTGAGT CAATGATAGTGCCGTTTACTAAGAAGAAACCAAGGAAAAAATTTGGGGTGCAGGGATCAAAACTTTTTGG AACATATGAAAGTACGTGTTTATACTCTTTATGGCCCTTGTCACTATGTATGCCTCGCTGCCTCCATTGG ACTCTAGAATGAAGCCAGGCAAGAGCAGGGTCTATGTGTGATGGCACATGTGGCCAGGGTCATGCAACAT GTACTTTGTACAAACAGTGTATATTGAGTAAATAGAAATGGTGTCCAGGAGCCGAGGTATCGGTCCTGCC AGGGCCAGGGGCTCTCCCTAGCAGGTGCTCATATGCTGTAAGTTCCCTCCAGATCTCTCCACAAGGAGGC ATGGAAAGGCTGTAGTTGTTCACCTGCCCAAGAACTAGGAGGTCTGGGGTGGGAGAGTCAGCCTGCTCTG GATGCTGAAAGAATGTCTGTTTTTCCTTTTAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTT GAAACAGGTAAGACAGGGGTCTAGCCTGGGTTTGCACAGGATTGCGGAAGTGATGAACCCGCAATAACCC TGCCTGGATGAGGGAGTGGGAAGAAATTAGTAGATGTGGGAATGAATGATGAGGAATGGAAACAGCGGTT CAAGACCTGCCCAGAGCTGGGTGGGGTCTCTCCTGAATCCCTCTCACCATCTCTGACTTTCCATTCTAAG CACTTTGAGGATGAGTTTCTAGCTTCAATAGACCAAGGACTCTCTCCTAGGCCTCTGTATTCCTTTCAAC AGCTCCACTGTCAAGAGAGCCAGAGAGAGCTTCTGGGTGGCCCAGCTGTGAAATTTCTGAGTCCCTTAGG GATAGCCCTAAACGAACCAGATCATCCTGAGGACAGCCAAGAGGTTTTGCCTTCTTTCAAGACAAGCAAC AGTACTCACATAGGCTGTGGGCAATGGTCCTGTCTCTCAAGAATCCCCTGCCACTCCTCACACCCACCCT GGGCCCATATTCATTTCCATTTGAGTTGTTCTTATTGAGTCATCCTTCCTGTGGTAGCGGAACTCACTAA GGGGCCCATCTGGACCCGAGGTATTGTGATGATAAATTCTGAGCACCTACCCCATCCCCAGAAGGGCTCA GAAATAAAATAAGAGCCAAGTCTAGTCGGTGTTTCCTGTCTTGAAACACAATACTGTTGGCCCTGGAAGA ATGCACAGAATCTGTTTGTAAGGGGATATGCACAGAAGCTGCAAGGGACAGGAGGTGCAGGAGCTGCAGG CCTCCCCCACCCAGCCTGCTCTGCCTTGGGGAAAACCGTGGGTGTGTCCTGCAGGCCATGCAGGCCTGGG ACATGCAAGCCCATAACCGCTGTGGCCTCTTGGTTTTACAGATACGAACCTAAACTTTCAAAACCTGTCA GTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGT CCAGCTGAGGTGAGGGGCCTTGAAGCTGGGAGTGGGGTTTAGGGACGCGGGTCTCTGGGTGCATCCTAAG CTCTGAGAGCAAACCTCCCTGCAGGGTCTTGCTTTTAAGTCCAAAGCCTGAGCCCACCAAACTCTCCTAC TTCTTCCTGTTACAAATTCCTCTTGTGCAATAATAATGGCCTGAAACGCTGTAAAATATCCTCATTTCAG CCGCCTCAGTTGCACTTCTCCCCTATGAGGTAGGAAGAACAGTTGTTTAGAAACGAAGAAACTGAGGCCC CACAGCTAATGAGTGGAGGAAGAGAGACACTTGTGTACACCACATGCCTTGTGTTGTACTTCTCTCACCG TGTAACCTCCTCATGTCCTCTCTCCCCAGTACGGCTCTCTTAGCTCAGTAGAAAGAAGACATTACACTCA TATTACACCCCAATCCTGGCTAGAGTCTCCGCACCCTCCTCCCCCAGGGTCCCCAGTCGTCTTGCTGACA ACTGCATCCTGTTCCATCACCATCAAAAAAAAACTCCAGGCTGGGTGCGGGGGCTCACACCTGTAATCCC AGCACTTTGGGAGGCAGAGGCAGGAGGAGCACAGGAGCTGGAGACCAGCCTGGGCAACACAGGGAGACCC CGCCTCTACAAAAAGTGAAAAAATTAACCAGGTGTGGTGCTGCACACCTGTAGTCCCAGCTACTTAAGAG GCTGAGATGGGAGGATCGCTTGAGCCCTGGAATGTTGAGGCTACAATGAGCTGTGATTGCGTCACTGCAC TCCAGCCTGGAAGACAAAGCAAGATCCTGTCTCAAATAATAAAAAAAATAAGAACTCCAGGGTACATTTG CTCCTAGAACTCTACCACATAGCCCCAAACAGAGCCATCACCATCACATCCCTAACAGTCCTGGGTCTTC CTCAGTGTCCAGCCTGACTTCTGTTCTTCCTCATTCCAGATCTGCAAGATTGTAAGACAGCCTGTGCTCC CTCGCTCCTTCCTCTGCATTGCCCCTCTTCTCCCTCTCCAAACAGAGGGAACTCTCCTACCCCCAAGGAG GTGAAAGCTGCTACCACCTCTGTGCCCCCCCGGCAATGCCACCAACTGGATCCTACCCGAATTTATGATT AAGATTGCTGAAGAGCTGCCAAACACTGCTGCCACCCCCTCTGTTCCCTTATTGCTGCTTGTCACTGCCT GACATTCACGGCAGAGGCAAGGCTGCTGCAGCCTCCCCTGGCTGTGCACATTCCCTCCTGCTCCCCAGAG ACTGCCTCCGCCATCCCACAGATGATGGATCTTCAGTGGGTTCTCTTGGGCTCTAGGTCCTGCAGAATGT TGTGAGGGGTTTATTTTTTTTTAATAGTGTTCATAAAGAAATACATAGTATTCTTCTTCTCAAGACGTGG GGGGAAATTATCTCATTATCGAGGCCCTGCTATGCTGTGTATCTGGGCGTGTTGTATGTCCTGCTGCCGA TGCCTTC f SEQ ID NO: 43 ]

[0296] In certain embodiments, the first antigen binding chain or the second antigen binding chain comprises a constant domain comprising a native or modified TRBC polypeptide. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of 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: 44 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44. SEQ ID NO: 44 is provided below.

[0297] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALND SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQG VLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 44 ]

[0298] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 44 is set forth in SEQ ID NO: 45, which is provided below.

[0299] GATCTGAAAAACGTGTTCCCTCCTGAAGTGGCTGTCTTTGAACCATCCGAGGCCGAGATTTCCCATACCC AGAAAGCAACTCTGGTCTGTCTGGCCACTGGATTCTACCCCGATCACGTGGAACTGTCTTGGTGGGTGAA CGGCAAGGAAGTCCATTCCGGAGTCTCTACCGACCCTCAGCCCCTCAAGGAGCAGCCTGCTCTCAACGAT TCTCGGTACTGCCTGTCATCTCGACTGAGAGTGTCTGCCACCTTCTGGCAGAACCCTAGAAACCACTTTC GGTGTCAGGTCCAGTTTTACGGCCTGAGCGAGAACGATGAGTGGACACAGGATAGAGCCAAACCTGTGAC ACAGATTGTGAGCGCCGAGGCTTGGGGACGAGCCGATTGTGGCTTCACATCCGAGTCTTACCAGCAGGGA GTGCTGTCTGCTACAATCCTCTACGAAATTCTCCTGGGGAAGGCCACCCTGTACGCTGTCCTCGTGTCTG CTCTGGTGCTCATGGCTATGGTCAAACGAAAGGACTCTAGAGGC [SEQ ID NO: 45 ]

[0300] In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of 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: 46 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 46. SEQ ID NO: 46 is provided below.

[0301] LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQ QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 46]

[0302] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 46 is set forth in SEQ ID NO: 47, which is provided below. CTGGAGGATCTGAAAAACGTGTTCCCTCCTGAAGTGGCTGTCTTTGAACCATCCGAGGCCGAGATTTCCC ATACCCAGAAAGCAACTCTGGTCTGTCTGGCCACTGGATTCTACCCCGATCACGTGGAACTGTCTTGGTG GGTGAACGGCAAGGAAGTCCATTCCGGAGTCTCTACCGACCCTCAGCCCCTCAAGGAGCAGCCTGCTCTC AACGATTCTCGGTACTGCCTGTCATCTCGACTGAGAGTGTCTGCCACCTTCTGGCAGAACCCTAGAAACC ACTTTCGGTGTCAGGTCCAGTTTTACGGCCTGAGCGAGAACGATGAGTGGACACAGGATAGAGCCAAACC TGTGACACAGATTGTGAGCGCCGAGGCTTGGGGACGAGCCGATTGTGGCTTCACATCCGAGTCTTACCAG CAGGGAGTGCTGTCTGCTACAATCCTCTACGAAATTCTCCTGGGGAAGGCCACCCTGTACGCTGTCCTCG TGTCTGCTCTGGTGCTCATGGCTATGGTCAAACGAAAGGACTCTAGAGGC [SEQ ID NO: 47 ] In certain embodiments, the TRBC polypeptide is a TRBC1 polypeptide. In certain embodiments, the TRBC1 polypeptide comprises or consists of 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: 48 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 48. SEQ ID NO: 48 is provided below.

[0303] LNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDS RYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGV LSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 48 ]

[0304] In certain embodiments, the TRBC1 polypeptide comprises or consists of 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: 49 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49. SEQ ID NO: 49 is provided below.

[0305] DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALND SRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQG VLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 49]

[0306] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49 is set forth in SEQ ID NO: 50, which is provided below.

[0307] GACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCC AAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAA TGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGAC TCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCC GCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCAC CCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGG GTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCG CCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC [SEQ ID NO: 50 ]

[0308] In certain embodiments, the TRBC polypeptide comprises or consists of 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 647196 (TRBC1, SEQ ID NO: 51), NCBI Genbank ID: 28638, NG_001333.2 range 655095 to 656583 (TRBC2, SEQ ID NO: 52) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 51. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 52. SEQ ID NO: 51 and 52 are provided below.

[0309] AGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACAC CCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTG AATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATG ACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTT CCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTC ACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGGTGAGTGGGGCCTGGGGAGATGCCTGGAGGAGA TTAGGTGAGACCAGCTACCAGGGAAAATGGAAAGATCCAGGTAGCAGACAAGACTAGATCCAAAAAGAAA GGAACCAGCGCACACCATGAAGGAGAATTGGGCACCTGTGGTTCATTCTTCTCCCAGATTCTCAGCCCAA CAGAGCCAAGCAGCTGGGTCCCCTTTCTATGTGGCCTGTGTAACTCTCATCTGGGTGGTGCCCCCCATCC CCCTCAGTGCTGCCACATGCCATGGATTGCAAGGACAATGTGGCTGACATCTGCATGGCAGAAGAAAGGA GGTGCTGGGCTGTCAGAGGAAGCTGGTCTGGGCCTGGGAGTCTGTGCCAACTGCAAATCTGACTTTACTT TTAATTGCCTATGAAAATAAGGTCTCTCATTTATTTTCCTCTCCCTGCTTTCTTTCAGACTGTGGCTTTA CCTCGGGTAAGTAAGCCCTTCCTTTTCCTCTCCCTCTCTCATGGTTCTTGACCTAGAACCAAGGCATGAA GAACTCACAGACACTGGAGGGTGGAGGGTGGGAGAGACCAGAGCTACCTGTGCACAGGTACCCACCTGTC CTTCCTCCGTGCCAACAGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCT AGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTAAGCAGGAGGGCA GGATGGGGCCAGCAGGCTGGAGGTGACACACTGACACCAAGCACCCAGAAGTATAGAGTCCCTGCCAGGA TTGGAGCTGGGCAGTAGGGAGGGAAGAGATTTCATTCAGGTGCCTCAGAAGATAACTTGCACCTCTGTAG GATCACAGTGGAAGGGTCATGCTGGGAAGGAGAAGCTGGAGTCACCAGAAAACCCAATGGATGTTGTGAT GAGCCTTACTATTTGTGTGGTCAATGGGCCCTACTACTTTCTCTCAATCCTCACAACTCCTGGCTCTTAA TAACCCCCAAAACTTTCTCTTCTGCAGGTCAAGAGAAAGGATTTCTGA [SEQ ID NO: 51 ] AGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACAC CCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTG AATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATG ACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTT CCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTC ACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGGTGAGTGGGGCCTGGGGAGATGCCTGGAGGAGA TTAGGTGAGACCAGCTACCAGGGAAAATGGAAAGATCCAGGTAGCGGACAAGACTAGATCCAGAAGAAAG CCAGAGTGGACAAGGTGGGATGATCAAGGTTCACAGGGTCAGCAAAGCACGGTGTGCACTTCCCCCACCA AGAAGCATAGAGGCTGAATGGAGCACCTCAAGCTCATTCTTCCTTCAGATCCTGACACCTTAGAGCTAAG CTTTCAAGTCTCCCTGAGGACCAGCCATACAGCTCAGCATCTGAGTGGTGTGCATCCCATTCTCTTCTGG GGTCCTGGTTTCCTAAGATCATAGTGACCACTTCGCTGGCACTGGAGCAGCATGAGGGAGACAGAACCAG GGCTATCAAAGGAGGCTGACTTTGTACTATCTGATATGCATGTGTTTGTGGCCTGTGAGTCTGTGATGTA AGGCTCAATGTCCTTACAAAGCAGCATTCTCTCATCCATTTTTCTTCCCCTGTTTTCTTTCAGACTGTGG CTTCACCTCCGGTAAGTGAGTCTCTCCTTTTTCTCTCTATCTTTCGCCGTCTCTGCTCTCGAACCAGGGC ATGGAGAATCCACGGACACAGGGGCGTGAGGGAGGCCAGAGCCACCTGTGCACAGGTGCCTACATGCTCT GTTCTTGTCAACAGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGG AAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTAAGGAGGAGGGTGGGAT AGGGCAGATGATGGGGGCAGGGGATGGAACATCACACATGGGCATAAAGGAATCTCAGAGCCAGAGCACA GCCTAATATATCCTATCACCTCAATGAAACCATAATGAAGCCAGACTGGGGAGAAAATGCAGGGAATATC ACAGAATGCATCATGGGAGGATGGAGACAACCAGCGAGCCCTACTCAAATTAGGCCTCAGAGCCCGCCTC CCCTGCCCTACTCCTGCTGTGCCATAGCCCCTGAAACCCTGAAAATGTTCTCTCTTCCACAGGTCAAGAG AAAGGATTCCAGAGGCTAG [SEQ ID NO: 52 ]

[0310] In certain embodiments, the first antigen binding chain or the second antigen binding chain comprises a constant domain comprising a native or modified TRGC polypeptide. In certain embodiments, the TRGC polypeptide is a native or modified TRGC1 polypeptide. In certain embodiments, the TRGC1 polypeptide comprises or consists of 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: 53, which is provided below. In certain embodiments, the TRGC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53.

[0311] DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYM KFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYY MYLLLLLKSVVYFAI ITCCLLRRTAFCCNGEKS [SEQ ID NO: 53]

[0312] In certain embodiments, the TRGC polypeptide is a native or modified TRGC2 polypeptide. In certain embodiments, the TRGC2 polypeptide comprises or consists of 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: 54, which is provided below. In certain embodiments, the TRGC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54.

[0313] DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYM KFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKD ANDTLLLQLTNTSAYYTYLLLLLKSVVYFAI ITCCLLRRTAFCCNGEKS [SEQ ID NO: 54 ]

[0314] In certain embodiments, the TRGC polypeptide comprises or consists of 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: 55), NCBI Genbank ID: 6967, NG_001336.2, range 124376 to 133924 (TRGC2, SEQ ID NO: 56) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the TRGC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 55. In certain embodiments, the TRGC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 56. SEQ ID NO: 55 and 56 are provided below.

[0315] ATAAACAACTTGATGCAGATGTTTCCCCCAAGCCCACTATTTTTCTTCCTTCAATTGCTGAAACAAAGCT CCAGAAGGCTGGAACATACCTTTGTCTTCTTGAGAAATTTTTCCCTGATGTTATTAAGATACATTGGCAA GAAAAGAAGAGCAACACGATTCTGGGATCCCAGGAGGGGAACACCATGAAGACTAACGACACATACATGA AATTTAGCTGGTTAACGGTGCCAGAAAAGTCACTGGACAAAGAACACAGATGTATCGTCAGACATGAGAA TAATAAAAACGGAGTTGATCAAGAAATTATCTTTCCTCCAATAAAGACAGGTATGTGTTTACGCATATCA TCTGTCAGAACACTTCTTTGAAAGTGAATGCTGCATTTTTTCCTTTCAGTATTAATGAAAAACAAACATA AATCTTTCTTAAATATTGTTACATTTAATGGTAGCATAAATGCCCTGCTACTTTTCTATAGAATTAAAAT GGTATAGGTTTTGGAGAAAACAAAATTGAAAAAGTTACTGAAGGTTTGTCAGCCTCAGCTCCATTATCCA AAATAAGAAAGTCACGTGCTGGTTTTTAGGGTTGTTAGATGGATTAAAGAAACAACATACACAGAAGCAT CTAGCAACGTGACACGTGGTAAACGCTCAAAAAGTGTTCTCCCTTCTTTTGATGACTTTACTTGATCAGG AAATAACATATATATGTCTTTCAGGAATGTTCTGCCCAAGCAGGAGAGTCACTCACCTCAATCTTGCTAC CCACAAAGTTTAACCTAAAAACAACGGGTTCATTGTTGACAAAATGATGTTTATCTGTTGTTGACAGAAT GATGTTTATCTAAAAACAGTTCCAATTTTCTATTTCCTTTGCTGAGACACAAAGGGGAGGCAAATGTGCA AAGCTTGAGGGTAGTCTTACCACTGTGCTTAAGTGTTCTGATTTTTCTAGTGATCAGGGCAAAATAAAAA GTATAGTAAGTTCCAAGGCAGTGAATATTATACAGGAGAGAAGTTACAGTTTTATAATGTGTTTTCCTTT ACACTAAATTCTAAAAGTAAAAAGTCTTTTTTTTTTTTTGACAGAGTTTCACTCTTGTTGCCCAAGCAGG TGTGCTATGGTATGATCTCAGCTCACTGCAACCTCCACCTCCCGGGTTCAAGTGATTCTCTTACTTCAGC CTCCCGACAGGCTGGGATTGCAGGCGCCTGCCACCACACCTGGCTAATTTTTGTGTTTTTAGTAGAGATG GGGTTTCACCATGTTGGCCAGGCTGGTCTCAAATTCCTGACCTCAAGTGATCCATCCACCTCGGCCTCCA AGTGCTGGGATTATGGGCGTCAGCCACTGTGCCCAGCCTAAAAGTAAAATGTCTTTCATGAGCTTCCCAA GGCAGCTACGTTAAGGAGGACACTTCTCTTAATGTCATTCTACAGTAGATTTCTAATGCTCTTTCTTGGA AGTTTGTTTTTCTGAGAAAAGCTAAAAATATAACATGGAAGTGATCATATTATATAATCAATGAAGTGCT TTTCAAGGAGATAAAACTAATCTGGTCCACACTTGCAACCAACCTTGATTGAGAGAGAGAGAGAACTCAG GATACACTTGAAGATTTTATTATGGGGAACAGTTACTTTATTCTTTTTACCTCAATCAATGCATGGAAAT AAGTGATAGTCATTTTCATTTATCTTTTAATAAATGAAGTCACCATGAGGAAAATAAAAAGACATTGAAA ACCCATTAAAGTCAGCCCTTAAAGATATTTGGACATGCAGACTTGATAACTAACGTTTGCATTCTTGAGA CTTACCCAAAACCCATACCTCAAGTCCAAGTTTTTAGAATTCATGAAATAAAGATCTCAGTGAGTGCATA AAATTGCGCACCAGAATCATATCCGTATAGACAAGAACACATCTACTAGAAAAATAATAAACCAACACAC CAATGCAACTGTGTTTTCTTCTGTTTTAAAGTATGTTGTCTTTGTATGCATGTTTGCTTCTTCCTTTTTT TTTTTAACATCACAGATAAATTCAACTCTCACCTCAGGTTTTATTGAGAGAACTGTCAATGTGACTTGGC CTCTGTCTTTCTAGTCCCAGAAAGAATTGCACTGAAATCTGAGCTCCTGTAATAAAAACAACCATTTGCT GAGAGTAATTAACATACTGAAAGAGATTTTCTTAGAGTACACAATGGTGACATTATATTGCCTCTTTATA AATAACTTTCTATCTATTTCTGTGGATTATTCCTACAAAGTACTTTTCATATGTCCAATTTCTTTTCTTC CCCTACAACTACTGTCTGAATACTGGCTCTGCTATTTGCTGATATGATTCTCGGCAAGTTGCCTGCACTT TTTAAACTTTATTTCCTCATTCAGAACATGGGGCCATACATAATACAACTCACTTCAGTGTTATTGGGGA ATTAAACAAAAAATGCATGGGAAGCATTTAACATAGTGCCTGACACAATAATGAGTACTCAGTAGATGTT AGCTTTTATTAATATTGTTGTTGTTATGTCCAGAAACACTATACCTCCAGAAAATCATGGGTACTTGCTG GGGACATTGGGGATATGCATGATTTGGAAAAGAATGACTGCTTTTTTTGCTTAGATGAGAAATTTTTCTA AGCCAGACTCCTTCAAATATGTAAGATTCTGTTGTGGATTCAAGGACTGAAAGAATTCTTGGCCGAGTGT GGTGGCTTATCCCTGTAATCCCAGCATTTTGTGAGGACAAGGCAGGAAGATTGCTTGAGTCCAGGAGTTT GAAACCAGCCTGCGCAACATGGCGAAACCCTGTCTCTACAAAAAATACAAACATTAGCTCGGAGTGAGTG CTGACATGTGCCTGTACTCCCAGCTACTCAGAAGGCTGAGATGGGAGGATCTCATGAGCCTGGGGAGTTT GAGGCTTCAGTGAGCCGTGATGACACCGTACTATACTCCACTCCAGCCTGGGTGACAGTGAGACCCTGCC T C AAAAAAC AAAC AAAC AAAC AAAC AAAAC AAAAT T AAT CT T T T T GC T G AT GT C AT GT C AGC AGT GT GT G TTGAAGGCTGTAAAGCAGCCATTTGTTCAGTTTATTTTTCCATTGAACAAGTATTTATCAAAAACATACT TTGTGGCAGTCACTATGCTAGGAGCTATGAATACAGAAGGAAAAGTAAATGCTCTTGGATACTACACTCC AGTTGTGATAAAAAAGAAAAAATGTATTCTTCACCAACTTCAACATCTTGATGTGCAAAAACATAATACA TGAATTAGATCTACCTAATTACACAGAATTAGACCAATTGTTTCTGGAATTGTGGGCTCATATTTTTAAT AACTGTCCTCCTGCCTCTCTGTCGACAGGTTTTATAAATATTCATTTAATTACACACACACACACGAACA ATTGACTAGTACTTGCTCTCATTCTTCTAGATGTCATCACAATGGATCCCAAAGACAATTGTTCAAAAGA TGCAAATGGTAAGCTTTTGTGTTTTTCCCTTCCTCCTGATCATTTTGTTTTGAACTTCTCTGGCTTGAAA AATCAGGGAATGGATTTTGCTAGGTTGGATGCTGCAGAATGGACCTAGTGATATTTTAAATTAGTCCCTC ATTTTCTAGGAGTTGTATTAACAAACCTAACTACTGCTTTGGGGTATGAGATGACTGTAAATTAGAGAGG GTACAGTGGTATAGTGATATGCTTTTAATTATTTCAAAAAAAAGATTTTATTCATTCATGTGTCTTTTTT CTTTTTCTTTTCTTTTTTTTTTTTTTTTGGACAGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCGGTGGC AGTATCTCAGCTCACCACAACCTCCGCCTCCCGGCTTCAAGTGATTCTCCTGCCTCAGCTTCTCGAGTAG CTGGGACTACAGGCGCGTGCCACCATGCCCGGCTAATTTTTGTATTTTTAGTAGAGTTGGGGTTTCACCA TGTTGGCCAGGATGGCCTCGAATTTGTGACCTCGTGATCTGCCCCCTCGCCCTCCCGAACTGTTGGGATT ACAGGCGTGAGTCACTGTGCCCGGCCTCCTGTCCTGTCTTTTGTTTAATGACTGGGAAAAACATGATACC ATGTTGCTTCTCGAGTTGTTTTGTTTTAGTCTTTGGTCTTTGCTAGTAGCTAATAACACGAACTAGTGTT TATCAAGTGCTTTTTACACAGAAGGGCTTGGGCTGTGTTCTGCATTTTCTTGTTTAACCCTCTTAAAACT CCTATAAAATGGTACATATTTTTCTCCCAATTTACAGTCCCTTTAAAGCAAATAATTATAAAAATCCCTA TACATGTCACACAGCTAGATCTGGGATTTCAAATCAGGCCATCAAACAAAGAGTTTATGTACTTAGTAAG TTTTCTGTTCTTTTTCTACAATAGAGTCAGATAGCAAGAAATTACCAAGCCAGGAACCTGAAACAAAACG GACATCATGTGGGGCTGGGTGGGTGCATGGGCTTTGCAGACTGGACTTTCACTCCAGCTCTTTTAATGAT T AGGT GT AAGT GACC T AC AT T T T GT GAGC AAC AGT T T T C T C AT C AGO C AAC AAAG AAT AAT TAG ACC AG A TTCACAGTTATTGAAGAGATAAAGGCATGAATGTGAGATGTCTGGCATAGGGCATCTCATTTAGCAGACA CAGAATGAGTACTTGTTTCTGGCTTTTTCTCTCTACATATGCACAAAGAATGCGACTAGAAGCATGGGCT CTAGCCCTGCTCAACTTTCCTCTATTTCCAATACCAAGGGGCTCTGACTTAGGCTGCCACACCAGGCAAG GAGGGCAGTACCACCTCACTTGACCAAGGGCAGGGAGTCACGGACACATCACTTCTTGAGATCCTTTTCC ACACCAAGGACTGATGTTTCTGGAATTCTCACTTTATGAAGACAAAACATATAAATGGAAATTTTCTCAG GTAGAGACTCACTCTTGTAGCTCATTGAGTAGGCACTAGTGGTCCACCCCCACTGTCTTTACTTATTCCT TGACATCACATATCTCTTGCAAAACCTCAAATAATATTAAATGCAATCACCCAATAATAGCATAGCCATA ATTAGAGGCATTTAGGAAAGACAGGTGAGTGTGCCACAACTACCTAACACATCAGCAAATCTGGATTAAC CACTTTCTTTGATTTTCCACAATGCAACCTTACTTTTTAATAGTTGGGAATGTTCTAAGTGAATTTAGCA GAGGTTGTTAATCAACTTGAAAGCTGAATTCTGACTTGTCTGACTCTTGGTGGTGCTGGTAGCAGTAGAT GTTTACTTTTAGGTTTTGGTGGTGGTGGAATATCACTTCAACGTAAATCATCAGAAATAAGTATTTGTGA ACCCCTCTCGCATTAATGTATCTTATTCTGTAAAAAGAACATGTGCAATTTCTCTTAGATACACTACTGC TGCAGCTCACAAACACCTCTGCATATTACATGTACCTCCTCCTGCTCCTCAAGAGTGTGGTCTATTTTGC CATCATCACCTGCTGTCTGCTTAGAAGAACGGCTTTCTGCTGCAATGGAGAGAAATCATAA [SEQ ID NO: 55 ]

[0316] ATAAACAACTTGATGCAGATGTTTCCCCCAAGCCCACTATTTTTCTTCCTTCGATTGCTGAAACAAAACT CCAGAAGGCTGGAACATACCTTTGTCTTCTTGAGAAATTTTTCCCAGATATTATTAAGATACATTGGCAA GAAAAGAAGAGCAACACGATTCTGGGATCCCAGGAGGGGAACACCATGAAGACTAACGACACATACATGA AATTTAGCTGGTTAACGGTGCCAGAAGAGTCACTGGACAAAGAACACAGATGTATCGTCAGACATGAGAA TAATAAAAACGGAATTGATCAAGAAATTATCTTTCCTCCAATAAAGACAGGTATGTGTTTACACATATCA TCTGTCAGAACACTTCTTTGAAAGTGAATGCTGCATTTTTTCCTTTCAGTATTAATGAAAAACATAAATC TTTCTTAAAAATTGTTACATTTAATGGTAGCGTAAATGCCCTGCTACTTTTCTATAGAATTAAAATGGTA TAGGTTTTGGAGAAAACAAAATTGAAAAAGTTGCTGAAGGTTTGTCAGCCTCAGCTCCATTATCCAAAAT AAGAAAGTCACGTGCTGGTTTTTAGGGTTGTTAGATGGATTAAAGAAACAACATACACAGAAGCATCTAG CAACGTGACACGTGGTAAACGCTCAAAAAGTGTTCTCCCTTCTTTTGATGACTTTACTTGATCAGGAAAT AACATATATATGTCTTTCAGGAATGTTCTGCCCAAGCAGGAGAGTCACTCACCTCAATCTTGCTACCCAC AAAGTTTAACCTAAAAACAACGGGTTCATTGTTGACAAAATAATGTTTATCTGAAGATAACTGTAGATCA TATTTATCTGTAGATAATGTTTATCTGTGGAGTGTGGCTCTACAAAACATAGAATAGTCTTGGTCACTGC AGTTTTATAGAGGCCTTGGGTTTTTCAGAGTTTCATTTTATATATCACCATAAAGTAACATTTCATAATT ACAGGTTGGTAAGGCTTACATGTACAAACATTCTTCCATTTTCCATAATAAATGCATTTCCTGCCATTGG TGAATGCAGCTCAATAAACATTTATTGTACAATTATGACACGCCAGGCTTAGTGGAAATGTGGATGAACA GACAAGGATGAGTTACTGTCCTAAGGATGATGCATGACAGTGCAGAGAATATACTCTCTTCCTGATCACT CAGGGTCACTCATGATTCATGCGCGAGGTCCCAAAACAGTGCCTTTGATGCAGATTCTGTACATCTCTAG ACGATTGGTCCAAGGGCTGAATGTGCTCTGGCCCAGTGGTCCAGTCTGTCACTATATGTCAACATCCTGA ATATGAACATAACAGTCCAACATCTCAAGAGTGGGCATGAAAAGGACTCATTTTGTGCTTTTTCCTGTGG TTAACAAGTCCTTTTTAGCCTGGGGGAACAAGCATTAACAAAATGTTTGAAGATCTTTGCCACGTACCAT TCCAAATTTCTAGGGTAAGTCTTTAGCTTTTCAGATCCTGAGTTTCTGCAATGATCAAATGTGATTTGGA CAGTTGCGTTGACTTTCTCCTGGGGCTATAATGGAGTGCAAAGGAAACAATGGCAGGGAAAATGCTTGCT TTCAAAATGGTAGCATGGATGTGTTCATTCGTGTAGTTACTGTATTAGGTATAGCCTTTCCTGAAACTAA CTGAAGTGGGGTTATAAAAACAGTCCCAATTTTCTATTTCCTTTGCTGAGACACAAAGAGGAGACAAAAG AGCAAAGCTTGAGGGTAGTTTTACCACTGTGCTTAAGTGTTCTGATTTTTCCAGTGATCAGGGTGAAATA AAAAGCATAGTAAGTTCCAGGGCAGTGAATACCATACAGGAGACAAGTTACAGTTTTATAATGTGTTTTA CTTTACACTAAATTCTAAAAGTAAAATGTCTTTTTTTTTTTCCGAGACAGAGTTTCACTCTTGTAGCCCA GGCAGGAGTGCTATGGTGTGATCTCGGCTCACAGCAACCTCCACCTCCCAGTTTCAAGCGATTCTTCTGC CTCAGCCTCCCGAGAAGTTGAAATTACAGGTGCCTGGCACCATATCTCGCTAATTATTCTATTTTTAGTA GAGATCGGGTTTTACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACTTCAAGTGATCCACCCGCCTCAG CCTCCCAAAGTGCTGGGATTACAGGTGTGAGTCACTGTGCCGGACCTAACAGTAAAATGTCTTTCATGTG CTTCTCAAGGCAACTACATTAAGGAGGACACATCTCTTAATGTCATTCTACAGTAGATTTCTAATGCTCT TTCTTGGAAGTTTGTTTTTCTGAGAAGAGCTAAAAATATAATAACATGGAAGTGATCATATTATATAATC AATGAAGTGCTTTCAAAGGAGATAAAACTAACCTGGTCTGCATTTGCAACCAGCCTTGATTGAGAGAGAG AGAACTCAGGATACACTTAGAGATTTTATTATGGGGAATAGTTACTTTATTCATTTTACCTCAATCAATG CAT GG AAAT AAGT GAG AGT CAT T T T CAT T T AT C T T T T AAT AAAT AAAGT C ACC AT GAGG AAAAT G AAAAC CCATTAAAGTCAGTCCTTAAAGATATTTGGACATGCAGACATGATAACTAACATTTCCATTCGTGAGACT TACCCAAAACCTATACCTCAAGTCCATTTCTTAGAATACATGAAATAAAGATCTCAGTGAGTGTATAAAA CTGCACACCAGAATCATATCCGTATAGACAAGAATACATCTACTAGAAAAATATAAACCAAAACACCAAG GTGACTCTGTTTTTTTCTGTTTTAAAATATGTTGTCTTTGTATGCATGTTTGCTTCTTCCTTTTTTTTTT TAAACATCGCAGATAAATTCAACTCTCACCTCAGTTGAGAGAGAACTGTCAATGTGACTTGGCCTCTCTC TTTCTAGTCCCAGAAAGAATTGCACTGAAATGCTGAGCTCCTGTAATAAAAATGACCATTTGCTGAGAGT AATTAACATACTGAAAGAGATTTTCTTAGAATAGTGCACAATGGCCCAATGGTGACATTATATTGTCTCT TTATAAATTATTTTCTATCTATTTCTGTGGATTATTTCTACAAAGCACTTTTCATATGTCCAATTCCTTT TATTCCCCTACAAGTACTGACTGACTACTGGCTCTGCTGTTCACTGATATGACTTTCGGCAAGTTGCCTG CACTTTTTAAACGTTATTTCCTCATTCAGAACATGGGGCCATACAAAATACAACTCACTTCAGTGTTATT GGGGAATTAAACAAATAAATGCATGGGAAGCATTTAACATAGTGCCTGACACAATAATGAGCACTCAGTA GATGTTAGCTTTTATTAATATTGTTGTTGCTATGTCCAGAAACACTATACCTCCAGAAAATCATGGGTAC TTGCTGGGGACGTTGGGGATATGCATGATTTTGAAAGGAGTGACTGCTCTTTACTGCTCAGATGAGAAAT TTTTCTAAGCCAGACTCCTTCAAACATGTAAGATTCTGTTGTGGATTCTAGGACTGAAAGAATTCTTGGC CGAGTGTGGTGGCTTATCCTGGTAATCTCATCATTTGGGAGGACAAGGCAGGAAGATTGCTTGAGCCCAG GAGTTGGAAACAAGCCTGGACAACATGGCGAAACCCTGTCTCTACAAAAAATACAAACATTAGCTGGTCA TGGGAGTGAGTGCCTGTACTCCCAGCTACTCAGGAGGCTAAGATAGGAGGATCACCTGAGCCTGGGCAGT TTGAGGTTTCAGTGAGCCGTGATGACACCATACTATACTCCACTCCAGCCTGGGTGACAGTGACATCCTG CCTCAAAAAAACCCCCAAAATTATTCTTTTTGCTGATTTCATGTCAGCAGTGTGTGCTGAAGGCTGTAAA GTAGCCACTTGTTCTGTTTATTTTTCCATTGAACAAGTATTTATCAAAAACGTACTTTGTGGAAGGCACT GTGCTAGGAACTATGCATACAGAAGGAAAACCAAATGTTCTTGGATACTACACTCCAGTTGTGATAAAAA AGAAAAAAGTATTCTTCACAAACTTCAACATTTTGATGTGCAAAAACATAATATATGAATTAGATCTACC TAACTACACAGAATTAGACCAATTATTTCTGGGATTATGGGCTCATATTTTTAATAACTGTCCTCCTACC TCTCTGTT G AC AGGT T T T AT AAAT AT T CAT T T AAT T AC AC AC AGT C AC AG AC AC ACT CAGAC AC AC AC AC ATACACACACACACACACCTTGACAAATAATGGGCATGAACAATTGACTGGTACTTGCTCTCATTCTTCT AGATGTCACCACAGTGGATCCCAAATACAATTATTCAAAGGATGCAAATGGTAAGTTTTTGTGTTTTTTA TTTCCTCCTGATCATTTTAAGTTTTGAACTTCTCTGGCTTGAAAAATCAGGGAATGGATTTTGCTAGGTT GGATGCTGCAGAATGGACCTAATCATATTTTAAATTAGTCCCTCTTTTTCTAGGAGTTGTATTAACAAAC CTAACTACTGCTTCATGTAAGAGATGACTGTAAATTGAAGGGTACAGTGATATGCTTTCAGTTATTTCAA AAAACAGACTTTACTCATCCATGTGTCTTTTTTCTTTTCTTTTTTTTCTTTTTTGAGACGGAGTCTCGCT CTGTTGAACAGGCTGGATTGCAGTGACGCGATCTCACCTCACTACAACCTCCGCCTCTGGAGTTCAAGCG ATTCTCCAGCCTCAGCTTCTCAAGTAGCTGGGACTACAGGCACATGCCACCATGTCCGGGTCATCTTTGT ATTTTTAGCAGAGACCGGGTTTCACTATGTTGGCCAGGCTGGTCTAGAATTCCTGACTTCGTGATCTGCC CCCTCAGCCCTCCGAAGTGCTGGGATTACAGACGTGAGTCACTGTGCCCGGCCTAACAGTAAAATGTCTT TCATGCGCTTCTCAAGGCAACTACGTTAAGGAGGACACTTCTCTTAATGTCATTCTACAGTAGATTTCTA ATGCTCTTTCTTGGAAGTTTGTTTTTCTGAGAAAAGCTAAAAATATAACATGGAAGTGATCATATTGTAT AATCAATGAAGTGCTTTTCAAGGAGATAAAACTAATCTGGTCCACGTTTGCAACCAACCTTGATTGAGAG AGAGAGAGAACTCAGGATACACTTGGAGATTTTATTATGGGGAATAGTTACTTTATTCTTTTTTCCTCAA T C AAT T C AT GG AAAT AAGT GAT AGT CAT AT T CAT T T AT C T T T T AAT AAAT G AAGT C ACC AT GAGG AAAAT AAAAAGAC AT T GAAAAC CC AT T AAAGT T AGC CC T T AAAG AT AT T T GG AC AT GC AG AC T T GAT AAC T AAC G TTTGCATTCTTGAGACTTACCCAAAACCCATACCTCAAGTCCATGTTTTTAGAATTCATGAAATAAAGAT CTCAGTGAGTGCATAAAATTGCGCACCAGAATCATATCCGTATAGACAAGAACACATCTACTAGAAAAAT AATAAACCAACACACCAATGCAACTGTGTTTTCTTCTGTTTTAAAATATGTTGTCTTTGTATGCATGTTT GCTTCTTCCTTTTTTTTTTTTAACATCACAGATAAATTCAACTCTCACCTCAGGTTTTATTGAGAGAACT GTCAATGTGACTTGGCCTCTGTCTTTCTAGTCCCAGAAAGAATCGCACTGAAATGCTGAGCTCCTGTAAT AAAAATGACCATTTGCTGAGAGTAATTAACATACTGAAAGAGATTTTCTTAGAGTACACAATGGTGACAT TATATTGTCTCTTTATAAATAACTTTCTATCTATTTCTGTGGATTATTCCTACAAAGTACTTTTCATATG TCCAGTTTCTTTTCTTCCCCTACAACTACCGTCTGAATACTGGCTCTGCTATTTGCTGATATGATTCTCG GCAAGTTGCCTGCACTTTTTAAACTTTATTTCCTCATTCAGAACATGGGGCCATGTAATACTCATGTACG TGAGTATTACGTAATAATGCTCACTTAAGTGTTACTGGGGAATTAAACAAAAAAATGCATGGCAAGCATT TAACATAGTGCCTGACACAATAATGAGCACTCAGTAGATGTTAGATTTTATTAATATTGTTGTTGTTATG TCCGGAAACACTATACCTCCAGAAAATCATGGGTACTTGCTTGGGATGTTGGGGATATGCATGATTTGGA AAGGTATGACTGCTTTTTTCTGCTTAGATGAGAAATTTTTCTAAGCCAGACTCCTTCAAATATGTAAGAT TCTGTTGTGGATTCTAGGACGGAAAGAATTCTTGGTCAGGTGTGGTTTCTTATCCCTGTAATCCCAGAAT TTTGGGAGGACAAGGCAGGAAGATTGCTTGAGCCCAGGAGTTTGAAACCAGCCTGGGCAACAAGACGAAA CCCTGTCTCTACAAAAGTACATAAATTAGCTTGGCTTGGTGGTGTGTGCCTGTATTACCAGCTATTCGGG AGACTGAGATGGGAGGATCTCCTGAACCTGTGAAGTTTGAGGCTTCAGTGAGCCGTGATGACACCATACT ATACTCGACTCCAGCCTGTGCGACAGTGAGACTCTGCGTCAAAAAAAAAACCCCAAAATTATTGTTTTTG CTGATTTCAGGTCAGCAGTGTGTGCTGAAGGGTGTAAAGTAGCCACTTGATCAGTTTATTTTTCCACTGA ACAAGTATTTATCAAAAACATACTTTGTGGTCTGTTTTTGATAAATAAAAAGGCACTGTGCTAGGAGCCA TGAATACAGAAGGAAAACCAAATGTTCTTGGATACTACACTCCAGTTGTGATAAAAAAGAAAAATGTATT CTTCACGAACTTCAACATTTTGATATGCAAAAACATAGTATATAAATTAGATCTACCTGATTACGTAGAA TCAGACCAATTATTTCTGGAATTGAGGGCTCATATTTTTAATAACTGTCCTCCTGCCTCTCTGTTGACAG GTTTTATAAATATTCATTTAATTACACACACACACACACACACCTTGACAAATAATGGACATGAACAATT GACTAGTACTTGCTCTCATTCTTCTAGATGTCATCACAATGGATCCCAAAGACAATTGGTCAAAAGATGC AAATGGTAAGCTTTTGTGTTTTTCCTTTCCTCCTGATCATTTTAAGTTTTGAACTTCTCTGGCTTGAAAA ATCAGGGAATGGGCCGGGTGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGG ATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGGTGAAACCCCGTCTCTACTAAAAATACAAA AAATTAGCCGGGCTTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCG TGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCACTCCACTCCAGCCTGGGCGACA GAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAATCAGGGAATGGATTTTGCTAGG TTGGATGCTGCAGAATGGACCTAGTGATATTTTAAATTAGTCCCTCTTTTTCTAGGAGTTGTATTAACAA ACCTAACTACTGCTTCGGGTATGAGATGACTGTAAATTAGAGGGTACAGTGATATGCTTTCAGTTATTTC AAAAAACAGACTTTATTCATCCGTCTGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGGAGT CTCACTCTATCACCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACCATAACCTCCGCCTTACTGGTT CAAGCGATTCTCCAGCCTCAGCTTCTCAAGTAGCTGGGACTACAGGTGCACACCACCATACCTGGCTAAT TTTTGTATTTTTAATAGAGATGGGGTTTCACCACGCTGGCCAGGATGGTCTTGAATTCTTGACCTCGTGA TCTGCCCCCTCGGGCTCCCAAACTTCTGGGATTATAGGCGTGAGCCACTGTGCCCGGCCTTCTGTCTTTT GTTATAATGACTGGGGAAAACATGATACCATGTTGCTTCTTGAGTTGTTTTGTTTTAGTCTTTGGTCTTT GCTAGTAGCTAATAACACGAACTAGTGTTTATCAAGTGCTTTTTACACAGAAGGGCTTGTTCTGCATTTT CTAGTTTAATCATCTTAATACTCCTATAAAGTAGTACAATATATTTTCTCCCATTTTACAGTCCCTTTAA AGTAAATAACTATAAAAATCCCTTATACATGTCACACAGCTAGGTCTGGCATTTCAAATCAGGACATCAA ACAAAGAATTCGTGCAGTTACTAAGTCCTCTATTTTTTCTACAATAGAAAAAATAGCAAGAATTACAGAT AGCAAGACATTACAAGGCAGGAATCTGAAACGAAAGGGACATAATGTGGGGCTGGGTGGGTGCATGAGCT TTGCAGACTAGACTTTCATTCCAGCTCTTTTAATGATTAGGTGTAAGTGACCTACATTTTGTGAGTAACA GTTTTCTCATCAGCCAACTAAGAATAATTACACCAGATTCACAGTTATTGAAGAGATAAGGGCATGAATG TGAGATGTCTGGCGTAGGGTATCTCATTTAGCAGACACAGAATGAATACTTGTTTCTGGCTTTTTCTCTC TACATATGCACAAAGAATGTGACTAGAAGCATTGGCTCTAGCCCTGCTCAACTTTCCTCTATTTCCAATA CCAAGGGGCTCTGACTTAGGCTGCCACACCAGGCAAGGAGGGGCAGTACCACCTCACTTGACCAAGGGCA GGGAGTCACGGACACATCACTTCCTGAGATCCTTTTCCACACCAAGGACTGATGTTTCTGGAATTCTCAC TTTATGAAGACAAAACATATAAATGGAAATTTCTGCAGGAAGAGACTCACTCTTGTAGCTCATTGAGTAG GCACTAGTGGTCCACCCCCACTGTCTTTACTTATTCCTTGACATCACATATCTCTTGTAAAACCTCAAAT AATGTTAAATGCAATCACCCAATAATAGCATAGCCATAATTAGAGGCATTTAGGAAAGACAGGTGAGTGT GCCACAACTACCTAACACATCAGCAAATCTGGATTAACCACTTTCTTTGATTTTCCACAATGCAACCTTA CTTTTTAATAGTTGGGAATGTTCTAAGTGAATTTAGCAGAGGTTGTTAATCAACTTGAAAGCTGAATTCT GACTTGTCTGACTCTTGGTGGTGCTGGTAGCAGTAGATGTTTACTTTTAGGTTTTGGTGGTGGTGGAATA TCACTTCAACGTAAATCATCAGAAATAAGTATTTGTGAACCCCTCTCGCATTAATATATCTTATTCTGTA AAAAGAACATGTGCAATTTCTCTTAGATACACTACTGCTGCAGCTCACAAACACCTCTGCATATTACACG TACCTCCTCCTGCTCCTCAAGAGTGTGGTCTATTTTGCCATCATCACCTGCTGTCTGCTTAGAAGAACGG CTTTCTGCTGCAATGGAGAGAAATCATAA [SEQ ID NO: 56]

[0317] In certain embodiments, the first antigen binding chain or the second antigen binding chain comprises a constant domain comprising a native or modified TRDC polypeptide. In certain embodiments, the TRDC polypeptide comprises or consists of 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: 57, which is provided below. In certain embodiments, the TRDC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

[0318] SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVT CSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTV AVNFLLTAKLFFL [SEQ ID NO: 57 ]

[0319] In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region that links the first antigen binding chain to the constant domain. In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region that links the second antigen binding chain to the constant domain. The hinge / spacer region can be flexible enough to allow the antigen binding chain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region can be the hinge region from IgGl, the CH2CH3 region of immunoglobulin and portions of CD3, a portion of a TCRa polypeptide, a portion of a TCRP polypeptide, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, or a synthetic spacer sequence. In certain embodiments, the hinge / spacer region comprises a portion of a TCRa polypeptide. In certain embodiments, the hinge / spacer region comprises a portion of the variable region (TRAV), a portion of the diversity region (TRAD), a portion of the joining region (TRAJ), a portion of the constant region (TRAC), or a combination thereof. In certain embodiments, the hinge / spacer region comprises a portion of the TRAJ region and a portion of the TRAC region of the TCRa polypeptide. In certain embodiments, the hinge / spacer region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, the hinge / spacer region comprises or consists of amino acids 1 to 3 of the sequence set forth in SEQ ID NO: 58. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 58 is set forth in SEQ ID NO: 59. SEQ ID NO: 58 and 59 are provided below.

[0320] IPNIQNPDPA [SEQ ID NO: 58 ]

[0321] ATTCCCAATATCCAGAACCCTGACCCTGCC [SEQ ID NO: 59]

[0322] In certain embodiments, the hinge / spacer region comprises a portion of a TCRP polypeptide. In certain embodiments, the hinge / spacer region comprises a portion of the variable region (TRBV), a portion of the diversity region (TRBD), a portion of the joining region (TRBJ), a portion of the constant region (TRBC), or a combination thereof. In certain embodiments, the hinge / spacer region comprises a portion of the TRBJ region and a portion of the TRAC region (C) of the TCRP polypeptide. In certain embodiments, the hinge / spacer region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the hinge / spacer region comprises or consists of amino acid 1 to 2 of the sequence set forth in SEQ ID NO: 60. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 60 is set forth in SEQ ID NO: 61. SEQ ID NO: 60 and 61 are provided below.

[0323] LEDLKNVFPPE [SEQ ID NO: 60]

[0324] CTGGAGGATCTGAAAAACGTGTTCCCTCCTGAA [SEQ ID NO: 61]

[0325] In certain embodiments, the antigen binding chain does not comprise an intracellular domain. In certain embodiments, the antigen binding chain is capable of associating with a CD3(^ polypeptide. In certain embodiments, the antigen binding chain associating with the CD3(^ polypeptide via the constant domain. 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 a target antigen is capable of activating the CD3(^ polypeptide associated to the antigen binding chain. In certain embodiments, the exogenous CD3(^ polypeptide is fused to or integrated with a costimulatory molecule disclosed herein.

[0326] In certain embodiments, the TCR-like fusion molecule comprises an antigen binding chain that comprises an intracellular domain.

[0327] In certain embodiments, the intracellular domain comprises a pTCR polypeptide. In certain embodiments, binding of the antigen binding chain to an antigen is capable of activating the pTCR polypeptide of the antigen binding chain. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 281 of SEQ ID NO: 16. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 233 of SEQ ID NO: 16. In certain embodiments, the intracellular domain comprises a CD3(^ polypeptide. In certain embodiments, binding of the antigen binding chain to an antigen is capable of activating the CD3(^ polypeptide of the antigen binding chain.

[0328] In certain embodiments, the CD3(^ polypeptide comprises or consists of 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 to the amino acid sequence set forth in SEQ ID NO: 12 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3(^ polypeptide comprises or consists of an amino acid sequence that is a consecutive portion of SEQ ID NO: 20, which is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, the CD3(^ comprises or consists of the amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 20. In certain embodiments, the CD3(^ polypeptide comprises or consists of amino acids 52 to 164 of SEQ ID NO: 20.

[0329] In certain embodiments, the CD3(^ polypeptide comprises or consists of 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 a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD3(^ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

[0330] In certain embodiments, the TCR-like fusion molecule comprises an antigen binding chain that comprises an intracellular domain, wherein the intracellular domain comprises a pTCR polypeptide. In certain embodiments, the intracellular domain comprises a pTCR polypeptide and a CD3(^ polypeptide. In certain embodiments, the intracellular domain comprises a pTCR polypeptide and does not comprise a CD3(^ polypeptide. In certain embodiments, the costimulatory signaling region comprises at least an intracellular domain of a co-stimulatory molecule disclosed herein.

[0331] In certain embodiments, the TCR-like fusion molecule comprises an antigen binding chain that comprises an intracellular domain, wherein the intracellular domain comprises a costimulatory signaling region. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and a CD3(^ polypeptide. In certain embodiments, the intracellular domain comprises a co-stimulatory signaling region and a pTCR polypeptide. In certain embodiments, the intracellular domain comprises a co-stimulatory signaling region and a pTCR polypeptide and does not comprise a CD3(^ polypeptide. In certain embodiments, the co- stimulatory signaling region comprises at least an intracellular domain of a co-stimulatory molecule disclosed herein.

[0332] In certain embodiments, the TCR-like fusion molecule is capable of associating with a CD3 complex (also known as “T-cell co-receptor”). In certain embodiments, the TCR-like fusion molecule 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 TCR-like fusion molecule replaces a native and / or an endogenous TCR in the CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3y chain, a CD36 chain, and two CD3s chains.

[0333] In certain embodiments, the CD3y chain comprises or consists of 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 amino acid sequence having a NCBI reference number: NP 000064.1 (SEQ ID NO: 62) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 62 is provided below.

[0334] MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKK KWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQD GVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN [SEQ ID NO: 62 ]

[0335] In certain embodiments, the CD36 chain comprises or consists of 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 amino acid sequence having a NCBI reference numbers: NP 000723.1 (SEQ ID NO: 63) or a fragment thereof, or the amino acid sequence having an NCBI reference number: NP 001035741.1 (SEQ ID NO: 64) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 63 and 64 are provided below.

[0336] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGI YRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGI IVTDVIATLLLALGVFCFAGHETGRLSGAADTQ ALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 63] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGI YRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 64 ]

[0337] In certain embodiments, the CD3s chain comprises or consists of 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 amino acid sequence having a NCBI reference number: NP 000724.1 (SEQ ID NO: 65) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. SEQ ID NO: 65 is provided below.

[0338] MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDE DDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICI TGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

[0339] [SEQ ID NO: 65 ]

[0340] In certain embodiments, the TCR-like fusion molecule exhibits a greater antigen sensitivity than a CAR targeting the same antigen. In certain embodiments, the TCR-like fusion molecule is capable of inducing an immune response when binding to an antigen that has a low antigen density on the surface of a tumor cell. In certain embodiments, cells comprising the TCR-like fusion molecule can be used to treat a subject having tumor cells with a low expression level of a 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 a low antigen density of a target molecule on the surface of the tumor cells. In certain embodiments, a target molecule having a low antigen 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 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 antigen 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 antigen 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 antigen 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 antigen 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 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 per cell and about 100 molecules per cell.

[0341] In certain embodiments, the TCR-like fusion molecule is capable of inducing an immune response when binding to an antigen that is expressed on the surface of a tumor cell having a low tumor cell frequency. In certain embodiments, cells comprising the TCR-like fusion molecule can be used to treat a subject having tumor cells with a low tumor cell frequency, e.g., from a relapse of a disease, wherein the subject received treatment which leads to residual tumor cells. In certain embodiments, the tumor having a low tumor cell frequency has a frequency that is less than about 40% per tumor, less than about 30% per tumor, less than about 20% per tumor, less than about 15% per tumor, less than about 10% per tumor, less than about 5% per tumor, less than about 2% per tumor, or less than about 1% per tumor. In certain embodiments, the low tumor cell frequency is less than about 2% per tumor. In certain embodiments, the low tumor cell frequency is less than about 1.5% per tumor. In certain embodiments, the low tumor cell frequency is less than about 1% per tumor. In certain embodiments, the low tumor cell frequency is between about 40% per tumor and about 20% per tumor, between about 20% per tumor and about 10% per tumor, between about 15% per tumor and about 10% per tumor, between about 20% per tumor and about 5% per tumor, between about 10% per tumor and about 2% per tumor, or between about 10% per tumor and about 1% per tumor.

[0342] In certain embodiments, the TCR-like fusion molecule comprises a first antigen binding chain comprising a VH of an antibody and a constant domain comprising a TRBC polypeptide; and a second antigen binding chain comprising a VL of an antibody and a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is designated as “VH-TRBC chain”. In certain embodiments, the second antigen binding chain is designated as “VL-TRAC chain”. In certain embodiments, the first antigen binding chain comprises a hinge region between the VH and the TRBC polypeptide. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or SEQ ID NO: 60. In certain embodiments, the second antigen binding chain comprises a hinge region between the VL and the TRAC polypeptide. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or SEQ ID NO: 60.

[0343] In certain embodiments, the TCR-like fusion molecule comprises a first antigen binding chain comprising a VH of an antibody and a constant domain comprising a TRAC polypeptide; and a second antigen binding chain comprising a VL of an antibody and a constant domain comprising a TRBC polypeptide. In certain embodiments, the first antigen binding chain is designated as “VH-TRAC chain”. In certain embodiments, the second antigen binding chain is designated as “VL-TRBC chain”. In certain embodiments, the first antigen binding chain comprises a hinge region between the VH and the TRAC polypeptide. In certain embodiments, the second antigen binding chain comprises a hinge region between the VL and the TRBC polypeptide. In certain embodiments, the first antigen binding chain and the second antigen binding chain bind to an antigen (e.g., human CD70).

[0344] In certain embodiments, the TCR-like fusion molecule that comprises a first antigen binding chain comprising a VH of an antibody and a constant domain comprising a TRBC polypeptide; and a second antigen binding chain comprising a VL of an antibody and a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is designated as “VH-TRBC chain”. In certain embodiments, the second antigen binding chain is designated as “VL-TRAC chain”. In certain embodiments, the first antigen binding chain comprises a hinge region between the VH and the TRAC polypeptide. In certain embodiments, the second antigen binding chain comprises a hinge region between the VL and the TRBC polypeptide. In certain embodiments, the first antigen binding chain and the second antigen binding chain bind to an antigen (e.g., human CD70).

[0345] 2.4.1. Exemplary TCR-like fusion molecules

[0346] In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen binding chains, e.g., a first antigen binding chain that comprises a VH and a TRBC polypeptide (“VH-TRBC chain”) and a second antigen binding chain that comprises a VL and a TRBC polypeptide (“VL-TRAC chain”), which are capable of dimerizing and binding to CD70. In certain embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 99, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 100, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 101. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 105. In certain embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 102, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 103, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 104. In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 106. In certain embodiments, the TRAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the TCR-like fusion molecule is designated as “70-HIT” or “70H”.

[0347] In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen binding chains, e.g., a first antigen binding chain that comprises a VH and a TRBC polypeptide (“VH-TRBC chain”) and a second antigen binding chain that comprises a VL and a TRBC polypeptide (“VL-TRAC chain”), which are capable of dimerizing and binding to CD70. In certain embodiments, the VH comprises a CDR1, a CDR2, and a CDR3 of a VH sequence of an anti-CD70 antibody disclosed in International Patent Publication No. WO 2007 / 038637, which is incorporated by reference in its entirety. In certain embodiments, the VH comprises a CDR1, a CDR2, and a CDR3 of a VH sequence of an anti-CD70 antibody 2H5 disclosed in International Patent Publication No. WO 2007 / 038637. In certain embodiments, the VL comprises a CDR1, a CDR2, and a CDR3 of a VH sequence of an anti-CD70 antibody disclosed in International Patent Publication No. WO 2007 / 038637. In certain embodiments, the VL comprises a CDR1, a CDR2, and a CDR3 of a VH sequence of an anti-CD70 antibody 2H5 disclosed in International Patent Publication No. WO 2007 / 038637.

[0348] In certain embodiments, the antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD19 (e.g., human CD19) and comprises two antigen binding chains, e.g., a first antigen binding chain that comprises a VH and a TRBC polypeptide (“VH-TRBC chain”) and a second antigen binding chain that comprises a VL and a TRBC polypeptide (“VL-TRAC chain”), which are capable of dimerizing and binding to CD19. In certain embodiments, the VH comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 107, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 108, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 109. In certain embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO: 113. In certain embodiments, the VL comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 110, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 111, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 112. In certain embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO: 114. In certain embodiments, the TRAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 41. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the TCR-like fusion molecule is designated as “19-HIT” or “19H”.

[0349] Various TCR-like fusion molecules are disclosed in International Patent Application Publication No. WO2019 / 133969, which is incorporated by reference hereby in its entirety.

[0350] 2.5. CCRs

[0351] In certain embodiments, the antigen-recognizing receptor is a CCR. The term “chimeric co-stimulating receptor” or “CCR” refers to a chimeric receptor that binds to an antigen and provides a co-stimulatory signal, but does not provide a T-cell activation signal to a cell comprising the CCR. Various CCRs are described in US20020018783 the contents of which are incorporated by reference in their entireties. CCRs mimic co-stimulatory signals, but unlike, CARs, do not provide a T-cell activation signal. In certain embodiments, the CCR lacks a CD3(^ polypeptide.

[0352] CCRs provide co-stimulation signal (e.g., a CD28-like signal or 4-lBB-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. Kloss et al., describe a strategy that integrates combinatorial antigen recognition, split signaling, and, critically, balanced strength of T-cell activation and co-stimulation 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 Biotechnology (2013);3 l(l):71-75, the content of which is incorporated by reference in 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 without rescue provided by simultaneous CCR recognition of the second antigen.

[0353] In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds to a second antigen and an intracellular domain that is capable of delivering a costimulatory signal to the cell but does not alone deliver an activation signal to the cell. In certain embodiments, the CCR further comprises a transmembrane domain.

[0354] In certain embodiments, the intracellular domain of the CCR comprises a pTCR polypeptide. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 281 of SEQ ID NO: 16. In certain embodiments, the pTCR polypeptide comprises or consists of amino acids 168 to 233 of SEQ ID NO: 16.

[0355] In certain embodiments, the intracellular domain of the CCR further comprises at least an intracellular domain of a co-stimulatory molecule or a portion thereof. In certain embodiments, the co-stimulatory molecule is selected from the group consisting of CD28, 4- IBB, 0X40, CD27, CD40, CD 154, CD97, CDlla / CD18, ICOS, DAP- 10, CD2, CD 150, CD226, and NKG2D.

[0356] In certain embodiments, the CCR comprises a pTCR polypeptide (e.g., amino acids 168 to 281 of SEQ ID NO: 16, amino acids 168 to 233 of SEQ ID NO: 16) and an intracellular domain of CD28 or a portion thereof. In certain embodiments, the CCR comprises a pTCR polypeptide (e.g., amino acids 168 to 281 of SEQ ID NO: 16, amino acids 168 to 233 of SEQ ID NO: 16) and an intracellular domain of 4- IBB or a portion thereof. In certain embodiments, the CCR comprises a pTCR polypeptide (e.g., amino acids 168 to 281 of SEQ ID NO: 16, amino acids 168 to 233 of SEQ ID NO: 16), an intracellular domain of CD28 or a portion thereof, and an intracellular domain of 4- IBB or a portion thereof.

[0357] In certain embodiments, the 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, LSCs, or AML HSPCs). Similar to a CAR, the extracellular antigen-binding domain can be an scFv, a Fab, an F(ab)2, or a chimeric protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, a cell can comprise a first antigen-recognizing receptor (e.g., a CAR, a TCR, or a TCR-like fusion molecule) and a CCR. In certain embodiments, a cell comprising a first antigen-recognizing receptor (e.g., a CAR, a TCR, or a TCR-like fusion molecule) and a CCR exhibits a greater degree of cytolytic activity against cells that are positive for both the first antigen and the second antigen as compared to against cells that are singly positive for the first antigen. In certain embodiments, the cell comprising the first antigenrecognizing receptor and the CCR exhibits substantially no or negligible cytolytic activity against cells that are singly positive for the first antigen.

[0358] In certain embodiments, the first antigen recognizing receptor binds to the first antigen with a low binding affinity, e.g., a dissociation constant (KD) of about 1 x 10'8M or more, about 5 x 10'8M or more, about 1 x 10'7M or more, about 5 x 10'7M or more, or about 1 x 10'6M or more, or from about 1 x 10'8M to about 1 x 10"6M. In certain embodiments, the antigen recognizing receptor (e.g., a CAR, a TCR, or a TCR-like fusion molecule) binds to the antigen with a low binding avidity. In certain embodiments, the antigen recognizing receptor (e.g., a CAR, a TCR, or a TCR-like fusion molecule) binds to the antigen at an epitope of low accessibility. In certain embodiments, the antigen recognizing receptor (e.g., a CAR, a TCR, or a TCR-like fusion molecule) binds to the antigen with a binding affinity that is lower compared to the binding affinity with which the CCR binds to the second antigen. In certain embodiments, the CCR binds to the third antigen with a binding affinity KD of from about 1 x 1 O’9M to about 1 x 1 O’7M, e.g., about 1 x 1 O’7M or less, about 1 x 10'8M or less, or about 1 x 10'9M or less.

[0359] 2.5.1. Exemplary CCRs

[0360] In certain embodiments, the antigen-recognizing receptor is a CCR that comprises (i) an extracellular antigen-binding domain, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising a pTCR polypeptide and a costimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co- stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the CAR is designated as “1 A-CCR.” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 126. SEQ ID NO: 126 is provided below.

[0361] FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSTC SCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPV [SEQ ID NO: 126]

[0362] In certain embodiments, the antigen-recognizing receptor is a CAR that comprises (i) an extracellular antigen-binding domain that binds CD 19, (ii) a transmembrane domain comprising a CD28 polypeptide, and (iii) an intracellular signaling domain comprising a pTCR polypeptide and a co-stimulatory signaling region comprising a CD28 polypeptide. In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153 to 179 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15. In certain embodiments, the intracellular signaling domain comprises, from N-end to C-end, (a) a co-stimulatory signaling region comprising a CD28 polypeptide that comprises or consists of 180 to 220 of SEQ ID NO: 15, and (b) a pTCR polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the CAR is designated as “19-1 A-CCR.” In certain embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 127. SEQ ID NO: 127 is provided below.

[0363] MALPVTALLLPLALLLHAEVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIY PGDGDTNYNGKFKGQATLT ADKS S STAYMQL SGLT SE DS AVY FCARKT I S S VVDFY FDY GQGTT VT VS S GGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRN SGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRAAAIEVMYPPPYLDNEK SNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSTCSCLCDPAGPLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQ PRDRRWGDTPPGRKPGSPV [SEQ ID NO: 127 ]

[0364] 3. Nucleic Acids and Vectors

[0365] The presently disclosed subject matter provides nucleic acids comprising a polynucleotide encoding an antigen-recognizing receptor comprising a pTCR polypeptide disclosed herein (e.g., disclosed in Section 2). Also provided are cells comprising such nucleic acids. In certain embodiments, the nucleic acid further comprises a first promoter that is operably linked to the polynucleotide encoding the antigen-recognizing receptor.

[0366] In certain embodiments, one or both of the first and second promoters are endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from an elongation factor (EF)-l promoter, a CMV promoter, a SV40 promoter, a PGK promoter, and a metallothionein promoter. In certain embodiments, one or both of the first and second promoters are inducible promoters. In certain embodiment, the inducible promoter is selected from a NFAT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter.

[0367] In certain embodiments, the antigen-recognizing receptors can be integrated at a locus within the genome of the T cell, e.g., a TRAC locus, a TRBC locus, a TRDC locus, or a TRGC locus. In certain embodiments, the locus is a TRAC locus. In certain embodiments, the expression of the antigen-recognizing receptors is under the control of an endogenous promoter. Nonlimiting examples of endogenous promoters include an endogenous TRAC promoter, an endogenous TRBC promoter, an endogenous TRDC promoter, and an endogenous TRGC promoter. In certain embodiments, the endogenous promoter is an endogenous TRAC promoter.

[0368] In certain embodiments, the nucleic acid is included in a vector. In certain embodiments, the vector is a retroviral vector (e.g., a gamma-retroviral vector or a lentiviral vector). In certain embodiments, the vector is viral vector selected from the group consisting of adenoviral vector, adena-associated viral vector, vaccinia virus, bovine papilloma virus, and herpes virus (e.g., such as Epstein-Barr Virus).

[0369] Additionally, the nucleic acid can be administered to subjects or and / delivered into cells by art-known methods or as described herein. Genetic modification of a cell (e.g., a T cell or a NK 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 nucleic acid compositions into the cell. For example, the polynucleotide 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.

[0370] The polynucleotide can be constructed in a single, multicistronic 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 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, picornavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A and F2A peptides). Combinations of retroviral vector and an appropriate packaging line are also suitable, where the capsid proteins will be functional for infecting human cells. Various amphotropic virus-producing cell lines are known, including, but not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437); PA317 (Miller, etal. (1986) Mol. Cell. Biol. 6:2895-2902); and CRIP (Danos, etal. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0371] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al. (1992) Blood 80:1418-1422, or 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, et al. (1992) J. Clin. Invest. 89:1817.

[0372] In certain embodiments, the delivery methods include use of colloids. As used herein, the term “colloid” refers to systems in which there are two or more phases, with one phase (e.g., the dispersed phase) distributed in the other phase (e.g., the continuous phase). Moreover, at least one of the phases has small dimensions (in the range of about 10-9to about 10-6m). Non-limiting examples of colloids encompassed by the presently disclosed subject matter include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems (e.g., micelles, liposomes, and lipid nanoparticles).

[0373] In certain embodiments, the delivery methods include use of liposomes. The term “liposome,” as used herein, refers to single- or multi-layered spherical lipid bilayer structures produced from lipids dissolved in organic solvents and then dispersed in aqueous media. Experimentally and therapeutically used for delivering an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein) to cells, liposomes fuse with cell membranes so the contents are transferred into the cytoplasm.

[0374] In certain embodiments, the delivery methods include use of lipid nanoparticles. As used herein, the term “lipid nanoparticle” refers to a particle having at least one dimension in the order of nanometers (e.g., from about 1 nm to about 1,000 nm) and including at least one lipid. In certain embodiments, the lipid nanoparticles can include an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein) for delivering to cells. The morphology of the lipid nanoparticles can be different from liposomes. While liposomes are characterized by a lipid bilayer surrounding a hydrophilic core, lipid nanoparticles have an electron-dense core where cationic lipids and / or ionizable lipids are organized into inverted micelles around an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein). Additional information on the morphology and properties of lipid nanoparticles and liposomes can be found in Wilczewska, et al., Pharmacological reports 64, no. 5 (2012): 1020-1037; Eygeris et al., Accounts of Chemical Research 55, no. 1 (2021): 2-12; Zhang et al., Chemical Reviews 121, no.

[0375] 20 (2021): 12181-12277; and Fan et al., Journal of pharmaceutical and biomedical analysis 192 (2021): 113642. In certain embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0376] In certain embodiments, the lipid nanoparticles can include a cationic lipid or an ionizable lipid. The term “cationic lipid” refers to lipids including a head group with permanent positive charges. Non-limiting examples of cationic lipids encompassed by the presently disclosed subject matter include l,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), l,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3 -di oleyloxy -N-[2-(sperminecarboxamido)ethyl]-N, N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), and ethylphosphatidylcholine (ePC).

[0377] As used herein, the term “ionizable lipid” refers to lipids that are protonated at low pH and are neutral at physiological pH. The pH-sensitivity of ionizable lipids is particularly beneficial for delivery in vivo (e.g., delivery of nucleic acid compositions disclosed herein), because neutral lipids have less interactions with the anionic membranes of blood cells and, thus, improve the biocompatibility of the lipid nanoparticles. Once trapped in endosomes, ionizable lipids are protonated and promote membrane destabilization to allow the endosomal escape of the nanoparticles. Non-limiting example of ionizable lipids encompassed by the presently disclosed subject matter include tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis (azanetriyl))tetrapropionate; decyl (2-(dioctylammonio)ethyl) phosphate; ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyl decanoate); bis(2- (dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate; l,l'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl) piperazin- l-yl)ethyl)azanediyl) bis(dodecan-2-ol); cKK-E12, 3, 6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5-dione; (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate; hexa(octan-3-yl) 9, 9', 9", 9"', 9'"', 9"'"- ((((benzene-l,3,5-tricarbonyl)yris(azanediyl)) tris (propane-3,1 -diyl)) tris(azanetriyl))hexanonanoate; heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino) octanoate; and (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4, 1-diyl))bis(azanetriyl))tetrakis(ethane-2,l-diyl) (9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)-tetrakis (octadeca-9, 12 - di enoate). Additionally, in certain embodiments, the lipid nanoparticles can include other lipids. For example, but without any limitation, the lipid nanoparticles of the presently disclosed subject matter can include phospholipids, cholesterol, polyethylene glycol (PEG)-functionalized lipids (PEG-lipids). These lipids can improve certain properties of the lipid nanoparticles (e.g., stability, biodistribution, etc.). For example, cholesterol enhances the stability of the lipid nanoparticles by modulating the integrity and rigidity. Non-limiting examples of other lipids present in lipid nanoparticles include cholesterol, DC-cholesterol, P-sitosterol, BHEM-cholesterol, ALC-0159, di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), di ol eoy Iphosphati dy 1 ethanol amine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl) -cyclohexane -1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE).

[0378] In certain embodiments, the lipid nanoparticles can include a targeting moiety that binds to a ligand. The use of the targeting moieties allows selective delivery of an active pharmaceutical ingredient (e.g., nucleic acid compositions disclosed herein) to target cells expressing the ligand (e.g., T cells). In certain embodiments, the targeting moiety can be an antibody or antigen-binding fragment thereof that binds to a cell surface receptor. For example, but without any limitation, the targeting domain is an antibody or antigen-binding fragment thereof that binds to a receptor expressed on the surface of a T cell (e.g., CD3, CD4, CD8, CD16, CD40L, CD95, FasL, CTLA-4, 0X40, GITR, LAG3, ICOS, and PD-1).

[0379] In certain embodiments, the delivery methods are in vivo delivery methods. In certain embodiments, the delivery methods are ex vivo delivery methods.

[0380] 4. Cells

[0381] The presently disclosed subject matter provides cells comprising the antigen-recognizing receptor comprising a pTCR polypeptide as disclosed in Section 2.

[0382] In certain embodiments, the cell is selected from the group consisting of cells of lymphoid lineage and cells of myeloid lineage. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the immunoresponsive cell is a cell of lymphoid lineage.

[0383] In certain embodiments, the cell is a cell of the lymphoid lineage. Cells of the lymphoid lineage can provide production of antibodies, regulation of cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, B cells, dendritic cells, stem cells from which lymphoid cells may be differentiated. In certain embodiments, the stem cell is a pluripotent stem cell (e.g., embryonic stem cell).

[0384] In certain embodiments, the cell is a T cell. 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 two types of effector memory T cells: e.g., TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocyte (TIL), Natural Killer T cells, Mucosal associated invariant T cells, and 76 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 introduction of an antigen-recognizing receptor, e.g., a CAR or a TCR. 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 CD8+T cell is CD4 independent. In certain embodiments, the T cell is derived from an induced pluripotent stem cell (iPSC). In certain embodiments, the T cell is a CD8+T cell that is CD4 independent, and the CD8+T cell is derived from an iPSC.

[0385] In certain embodiments, the T cell is a CD62L+T cell. In certain embodiments, the T cell is a CD45RA+T cell. In certain embodiments, the T cell is a CD62L+ / CD45RA+T cell.

[0386] In certain embodiments, the cell is a NK cell. Natural Killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0387] 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., et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-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., et al. 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 65:5417-5427; Papanicolaou, G. A., et al. 2003 Blood 102:2498-2505 (disclosing selectively in vitro-Q ^ n antigen-specific peripheral blood leukocytes employing artificial antigen-presenting cells (AAPCs) or pulsed dendritic cells).

[0388] In certain embodiments, the cell (e.g., T cell) is autologous. In certain embodiments, the cell (e.g., T cell) is non-autologous. In certain embodiments, the cell (e.g., T cell) is allogeneic. In certain embodiments, the cell (e.g., T cell) is derived in vitro from an engineered progenitor or stem cell.

[0389] In certain embodiments, the cell is a cell of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells from which myeloid cells may be differentiated.

[0390] In certain embodiments, the stem cell is a pluripotent stem cell (e.g., an embryonic stem cell or an induced pluripotent stem cell).

[0391] 4.1. T Cell Receptors (TCRs)

[0392] In certain embodiments, a presently disclosed cell comprising the antigen-recognizing receptor comprising a pTCR polypeptide further comprises a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with the invariant CD3 chain molecules. A TCR is found on the surface of T cells, and is responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, a TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, a TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0393] Each chain of a TCR is composed of two extracellular domains: Variable (V) region and a Constant (C) region. The Constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domain of both chains each has three complementarity determining regions (CDRs).

[0394] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules CD36 / s, CD3y / s and CD247

[0395]

[0396] or C / r|. When a TCR complex engages with its antigen and MHC (peptide / MHC), the T cell expressing the TCR complex is activated.

[0397] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the TCR is naturally occurring TCR.

[0398] In certain embodiments, the TCR is an exogenous TCR. In certain embodiments, the TCR is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, the non-naturally occurring TCR is modified from a naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100 or more amino acid residues.

[0399] 4.2. Co-stimulatory Ligands

[0400] In certain embodiments, a presently disclosed cell comprising the antigen-recognizing receptor comprising a pTCR polypeptide further comprises at least one recombinant or exogenous co-stimulatory ligand. For example, a presently disclosed cell can be further transduced with at least one co-stimulatory ligand, such that the cell expresses or is induced to express the first antigen-recognizing receptor, the second antigen-recognizing receptor, and the at least one co-stimulatory ligand. The at least one co-stimulatory ligand provides a co-stimulation signal to the cell.

[0401] Non-limiting examples of costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) 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 segment and a relatively long extracellular region. Non-limiting examples of TNF superfamily members include nerve growth factor (NGF), CD40L (also known as “CD 154”), 4-1BBL, TNF -a, OX40L, CD70, Fas ligand (FasL), CD30L, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell-activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins - they possess an immunoglobulin domain (fold). Non-limiting examples of immunoglobulin superfamily ligands include CD80, CD86, and ICOSLG. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, GITRL, CD40L, OX40L, CD30L, TNFRSF14, ICOSLG, TRAIL, and combinations thereof

[0402] In certain embodiments, the cell further comprises one exogenous co-stimulatory ligand that is 4-1BBL. In certain embodiments, the co-stimulatory ligand is human 4-1BBL. In certain embodiments, the 4-1BBL comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having a Uniprot Reference No: P41273-1 (SEQ ID NO: 66) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BBL comprises or consists of an amino acid sequence that is a consecutive portion of the amino acid sequence of SEQ ID NO: 66. SEQ ID NO: 66 is provided below.

[0403] MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRL REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGV YYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQ RLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE [SEQ ID NO: 66]

[0404] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 66 is set forth in SEQ ID NO: 67.

[0405] ATGGAATACGCCTCTGACGCTTCACTGGACCCCGAAGCCCCGTGGCCTCCCGCGCCCCGCGCTCGCGCCT GCCGCGTACTGCCTTGGGCCCTGGTCGCGGGGCTGCTGCTGCTGCTGCTGCTCGCTGCCGCCTGCGCCGT CTTCCTCGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTCCGCGGCCAGCCCGAGACTC CGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGC AGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGG CGTGTCCCTGACGGGGGGCCTGAGCTACAAAGAGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTC TACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTG CGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACC CGCCTCCTCCGAGGCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAG CGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCGCCACAG TCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAA [ SEQ ID NO: 67 ]

[0406] In certain embodiments, the cell further comprises one exogenous co-stimulatory ligand that is CD80. In certain embodiments, the co-stimulatory ligand is human CD80. In certain embodiments, the CD80 comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having a NCBI Reference No: NP 005182 (SEQ ID NO: 68) or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is a consecutive portion of the amino acid sequence of SEQ ID NO: 68. SEQ ID NO: 68 is provided below.

[0407] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEK KMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA DFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTT NHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERL RRESVRPV [SEQ ID NO: 68 ]

[0408] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 68 is set forth in SEQ ID NO: 69. SEQ ID NO: 69 is provided below.

[0409] ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTCTTGG TGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAAC GCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAG AAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTG ATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGT TGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCT GACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAA CCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACAC AACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACC AACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAA CCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAAT TTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTG AGAAGGGAAAGTGTACGCCCTGTA [SEQ ID NO: 69]

[0410] In certain embodiments, the cell further comprises two exogenous co-stimulatory ligands that are 4-1BBL and CD80. In certain embodiments, the cell further comprises two exogenous co-stimulatory ligands that are 4-1BBL and CD80, wherein the 4-1BBL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66, and the CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 68.

[0411] Receptor-comprising cells comprising at least one exogenous co-stimulatory ligand are described in U. S. Patent No. 8,389,282, which is incorporated by reference in its entirety.

[0412] 4.3. Fusion Polypeptides

[0413] In certain embodiments, a presently disclosed cell comprising the antigen-recognizing receptor comprising a pTCR polypeptide further comprises a fusion polypeptide. For example, a presently disclosed cell can be further transduced with the fusion polypeptide, such that the cell expresses or is induced to express the antigen-recognizing receptor comprising a pTCR polypeptide and the fusion polypeptide. The fusion polypeptide provides a co-stimulation signal to the cell. The fusion polypeptides are capable of enhancing the activity and / or efficacy of a cell comprising the first antigen-recognizing receptor (e.g., a CAR or a TCR-like fusion molecule). In certain embodiments, the fusion polypeptide comprises a) an extracellular domain and a transmembrane domain of a co-stimulatory ligand, and b) an intracellular domain of a first costimulatory molecule.

[0414] Non-limiting examples of the co-stimulatory ligand include tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. The TNF family member can be selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. The Ig superfamily member can be selected from the group consisting of CD80, CD86, ICOS ligand (ICOSLG (also known as “CD275”), and combinations thereof. In certain embodiments, the co-stimulatory ligand is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, CD80, CD86, ICOSLG, and combinations thereof.

[0415] In certain embodiments, the fusion polypeptide comprises an extracellular domain and a transmembrane domain of a co-stimulatory ligand that is CD80. In certain embodiments, the co-stimulatory ligand is human CD80. In certain embodiments, the CD80 comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 68 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is a consecutive portion of the amino acid sequence of SEQ ID NO: 68.

[0416] In certain embodiments, the extracellular domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 amino acids 1- 242 of SEQ ID NO: 68. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1-242 of SEQ ID NO: 68 or a functional fragment thereof. A functional fragment can be a consecutive portion of amino acids 1-242 of SEQ ID NO: 68, which is at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200, or at least about 220 amino acids in length. In certain embodiments, the functional fragment retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the extracellular domain of CD80. Non-limiting examples of the primary functions of the extracellular domain of CD80 include binding to / interacting with CD28, binding to / interacting with CTLA-4, binding to / interacting with PD-L1, and contributing to CD80 homodimerization. In certain embodiments, an extracellular domain of CD80 comprises or consists of amino acids 1-242 of SEQ ID NO: 68.

[0417] In certain embodiments, the transmembrane domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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 amino acids 243-263 of SEQ ID NO: 68. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243-263 of SEQ ID NO: 68 or a fragment thereof. Such fragment can be at least about 5, at least about 10, at least about 15, or at least about 20 amino acids in length. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243-263 of SEQ ID NO: 68.

[0418] In certain embodiments, the fusion polypeptide comprises an intracellular domain of a costimulatory molecule that is 4- IBB. In certain embodiments, the co-stimulatory molecule is human 4-1BB. In certain embodiments, the 4-1BB comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 38 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB comprises or consists of an amino acid sequence that is a consecutive portion of the amino acid sequence of SEQ ID NO: 38. In certain embodiments, the intracellular domain of 4- IBB comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to amino acids 214-255 of SEQ ID NO: 38 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of 4- IBB comprises or consists of amino acids 214-255 of SEQ ID NO: 38 or a functional fragment thereof. Such functional fragment can be a consecutive portion of amino acids 214-255 of SEQ ID NO: 70, which is at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 amino acids in length. In certain embodiments, the functional fragment of amino acids 214-255 of SEQ ID NO: 38 retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary functions of the intracellular domain of 4-1BB. Non-limiting examples of the primary functions of the intracellular domain of 4- IBB include providing costimulatory signaling for the activation and proliferation of an immunoresponsive cell (e.g., a T cell), and interacting and activating downstream adaptors (e.g., TRAFs). In certain embodiments, the intracellular domain of 4-1BB comprises or consists of amino acids 214-255 of SEQ ID NO: 38.

[0419] In certain embodiments, the co-stimulatory molecule is CD28. In certain embodiments, the co-stimulatory molecule is human CD28. In certain embodiments, the CD28 comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 15 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the CD28 comprises or consists of an amino acid sequence that is a consecutive portion of the amino acid sequence of SEQ ID NO: 15. In certain embodiments, the intracellular domain of CD28 comprises or consists of 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%, or at least about 99%, at least about 100% homologous or identical to amino acids 180 to 219 of SEQ ID NO: 15 or a fragment thereof, and / or may optionally comprise up to one or up to two or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180 to 219 of SEQ ID NO: 15 or a functional fragment thereof. A functional fragment of amino acids 180 to 219 of SEQ ID NO: 15 can be a consecutive portion of amino acids 180 to 219 of SEQ ID NO: 15, which is at least about 20, at least about 25, at least about 30, or at least about 35 amino acids in length. In certain embodiments, such functional fragment retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the intracellular domain of CD28. Non-limiting examples of the primary functions of the intracellular domain of CD28 include providing co-stimulatory signaling for the activation and proliferation of an immunoresponsive cell (e.g., a T cell), and interacting with protein adaptors (e.g., PI3K, GRB2, and LCK). In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180 to 219 of SEQ ID NO: 15.

[0420] In certain embodiments, the fusion polypeptide comprises an intracellular domain of a second co-stimulatory molecule. In certain embodiments, the fusion polypeptide comprises an intracellular domain of a third co-stimulatory molecule. In certain embodiments, the fusion polypeptide comprises an intracellular domain of a fourth co- stimulatory molecule. In certain embodiments, the fusion polypeptide comprises an intracellular domain of a fifth co-stimulatory molecule. In certain embodiments, the first, second, third, fourth, and fifth co-stimulatory molecule can be the same or different among each other.

[0421] In certain embodiments, the fusion polypeptide comprises an extracellular domain and a transmembrane domain of a co-stimulatory ligand that is CD80, and an intracellular domain of a co-stimulatory molecule that is 4-1BB. In certain embodiments, the fusion polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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: 70. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70. SEQ ID NO: 70 is provided below.

[0422] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEK KMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA DFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTT NHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFKRGRKKLLYIFKQ PFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 70]

[0423] In certain embodiments, the fusion polypeptide comprises an extracellular domain and a transmembrane domain of a co-stimulatory ligand that is CD80, an intracellular domain of a first co-stimulatory molecule that is 4- IBB, and an intracellular domain of a second co-stimulatory molecule that is CD28.

[0424] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, 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: 71. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71. SEQ ID NO: 71 is provided below.

[0425] MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEK KMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA DFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTT NHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFRSKRSRLLHSDYM NMTPRRPGPTRKHYQPYAPPRDFAAYRKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0426] [SEQ ID NO: 71]

[0427] Various modified fusion polypeptides are disclosed in International Patent Application No. PCT / US20 / 42753, which is incorporated by reference hereby in its entirety.

[0428] 4.4. Delivery of the Anti en-Re cognizing Receptor

[0429] In certain embodiments, the antigen-recognizing receptor is delivered to the cell by a viral method. In certain embodiments, the viral method comprises a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gammaretroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adeno-associated viral vectors, vaccinia viruses, bovine papilloma viruses, and herpes viruses (e.g., such as Epstein-Barr Virus).

[0430] In certain embodiments, the antigen-recognizing receptor is delivered to the cell by a non-viral method. Any targeted genome editing methods can also be used to deliver the antigenrecognizing receptor to the cell. In certain embodiments, the antigen-recognizing receptor is delivered to the cell by a method comprising homologous recombination, a Zinc finger nuclease, a meganuclease, a Transcription activator-like effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof. In certain embodiments, a CRISPR system is used to deliver the antigen-recognizing receptor to the cell.

[0431] In certain embodiments, the cell is a T cell, and the antigen-recognizing receptor is integrated at a locus within the genome of the T cell. Non-limiting examples of loci include a TRAC locus, a TRBC locus, a TRDC locus, and a TRGC locus. In certain embodiments, the locus is a TRAC locus or a TRBC locus. In certain embodiments, the cell is a T cell, and the antigenrecognizing receptor is integrated at a TRAC locus. Methods of targeting a CAR to a site within the genome of T cell are disclosed in WO2017180989 and Eyquem et al., Nature. (2017 Mar 2); 543(7643): 113-117, both of which are incorporated by reference in their entireties. In certain embodiments, the cell is a T cell, the first antigen-recognizing receptor is a CAR, and the first antigen-recognizing receptor is integrated at a TRAC locus. In certain embodiments, the cell further comprises a gene disruption of a TRBC locus. In certain embodiments, the gene disruption of a TRBC locus results in knockout of TRBC locus.

[0432] 4.5. Gene Disruptions and Gene Modifications

[0433] In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene disruption of a CD70 locus. The gene disruption of the CD70 locus can result in a non-functional CD70 protein or a knockout of the CD70 gene expression. In certain embodiments, the gene disruption of the CD70 locus results in knockout of the CD70 gene expression.

[0434] Non-limiting examples of gene disruptions include substitutions, deletions, insertions, or combinations thereof. In certain embodiments, the mutation comprises a missense mutation, a nonsense mutation, or a combination thereof. In certain embodiments, the deletion comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the insertion comprises a non-frameshift insertion, a frameshift insertion, or a combination thereof.

[0435] In certain embodiments, the CD70 locus is a human CD70 locus. The gene disruption of the CD70 locus can be generated by any suitable gene editing methods. In certain embodiments, the gene disruption of the CD70 locus (e.g., knockout of the CD70 locus) is generated using a viral method. In certain embodiments, the viral method comprises a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gammaretroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adena-associated viral vectors, vaccinia viruses, bovine papilloma viruses, and herpes viruses (e.g., such as Epstein-Barr Virus).

[0436] In certain embodiments, the gene disruption of the CD70 locus (e.g., knockout of the CD70 locus) is generated using a non-viral method. Non-viral approaches can also be employed for genetic modification of a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., PProc. 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., 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 microinjection 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 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 (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.

[0437] Any targeted genome editing methods can also be used to generate the gene disruption of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus is generated by a method comprising homologous recombination, a Zinc finger nuclease, a meganuclease, a Transcription activator-like effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0438] In certain embodiments, a CRISPR system is used to generate the gene disruption of the CD70 locus.

[0439] Clustered regularly-interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When utilized for genome editing, 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 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 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. In certain embodiments, the CRISPR system comprises base editors. In certain embodiments, the CRISPR system comprises transposases / recombinases. In certain embodiments, the CRISPR system comprises prime editors. In certain embodiments, the CRISPR system comprises an epigenetic modulator. In certain embodiments, the CRISPR system comprises is a CRISPRoff system. Additional details on the CRISPR systems of the presently disclosed subject matter can be found in Anzalone et al., Nature biotechnology 38.7 (2020): 824-844 and in Nunez et al., Cell 184.9 (2021): 2503-2519, the contents of each of which are incorporated by reference in their entireties.

[0440] In certain embodiments, the CD70 locus is disrupted using a gRNA molecule to knockout expression of CD70. The gRNA molecule can target a coding sequence of a CD70 gene (e.g., a human CD70 gene) or a non-coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets a coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets a target sequence within a human CD70 gene.

[0441] In certain embodiments, zinc-finger nucleases are used to generate the gene disruption of the CD70 locus. A zinc-finger nuclease (ZFN) is an artificial restriction enzyme, which is 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 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 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.

[0442] In certain embodiments, a TALEN system is used to generate the gene disruption of the CD70 locus. 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 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 (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 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.

[0443] Methods for delivering the genome editing agents / sy stems can vary depending on the need. In certain embodiments, the components of a selected genome editing method 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 elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic Nanoparticles, and cell-penetrating peptides).

[0444] In certain embodiments, the gene disruption of the CD70 locus can be a disruption of the coding region of the CD70 locus and / or a disruption of the non-coding region of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption of the coding region of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises an insertion at the coding region of the CD70 locus. Human CD70 protein comprises three exons: exon 1, exon 2, and exon 3. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption at one or more of exon 1, exon 2, and exon 3 of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption at exon 1 of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises an insertion at exon 1 of the CD70 locus.

[0445] In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene modification of a CD70 gene. The gene modification of the CD70 gene can result in a non-functional CD70 protein or a knockdown of the CD70 gene expression. In certain embodiments, the gene modification of the CD70 gene results in knockout of the CD70 gene expression.

[0446] In certain embodiments, the modification of the CD70 gene comprises use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent comprises a shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the CD70 gene and thereby reduces or eliminates the expression of the CD70 gene or CD70 protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses the first antigen-recognizing receptor and / or the second antigen-recognizing receptor disclosed herein. In certain embodiments, the expressions of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor are driven by identical promoters (e.g., a same promoter). In certain embodiments, the expressions of the shRNA, the first antigen-recognizing receptor, and the second antigen-recognizing receptor disclosed herein are driven by different promoters.

[0447] In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, 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 about 100% homologous or identical to at least a portion of a CD70 nucleic acid sequence. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to the CD70 gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is up to 15 nucleotides, up to 20 nucleotides, up to 25 nucleotides, up to 30 nucleotides, up to 35 nucleotides, up to 40 nucleotides, up to 55 nucleotides, up to 60 nucleotides, up to 65 nucleotides, up to 70 nucleotides, up to 75 nucleotides, up to 80 nucleotides, up to 85 nucleotides, up to 90 nucleotides, up to 95 nucleotides, or up to 100 nucleotides in length. In certain embodiments, the RNAi agent comprises DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0448] In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of CD70 by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% or any intermediate value or range thereof. In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of CD70 by about 70%.

[0449] In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene disruption of a TRAC locus. In certain embodiments, the gene disruption of the TRAC locus results in a non-functional TCR. In certain embodiments, the gene disruption of the TRAC locus results in knockout of the TCR gene expression.

[0450] Any methods to generate the gene disruption of the CD70 locus, as disclosed above, can be used to generate the gene disruption of the TRAC locus. In certain embodiments, the gene disruption of the TRAC locus is generated by a method comprising a gene editing method comprising homologous recombination, a Zinc finger nuclease, a meganuclease, a Transcription activator-like effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0451] In certain embodiments, the gene disruption of the TRAC locus can be a disruption of the coding region of the TRAC locus and / or a disruption of the non-coding region of the TRAC locus. In certain embodiments, the gene disruption of the TRAC locus comprises a disruption of the coding region of the TRAC locus. In certain embodiments, the gene disruption of the TRAC locus comprises an insertion at the coding region of the TRAC locus. Human TRAC protein comprises 4 exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRAC locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption of the TRAC locus comprises a disruption at one or more of exon 1 through exon 4 of the TRAC locus. In certain embodiments, the gene disruption of the TRAC locus comprises a disruption at exon 1 of the TRAC locus. In certain embodiments, the gene disruption of the TRAC locus comprises an insertion at exon 1 of the TRAC locus.

[0452] In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene modification of a TRAC gene. The gene modification of the TRAC gene can result in a non-functional TCR protein or a knockdown of the TCR gene expression. In certain embodiments, the gene modification of the TRAC gene results in knockout of the TCR gene expression.

[0453] In certain embodiments, the modification of the TRAC gene comprises use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent comprises a shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the TRAC gene and thereby reduces or eliminates the expression of the TRAC gene or TCR protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses the first antigen-recognizing receptor and / or the second antigen-recognizing receptor disclosed herein. In certain embodiments, the expressions of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor are driven by identical promoters (e.g., a same promoter). In certain embodiments, the expressions of the shRNA, the first antigen-recognizing receptor, and the second antigen-recognizing receptor disclosed herein are driven by different promoters.

[0454] In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, 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 about 100% homologous or identical to at least a portion of a TRAC nucleic acid sequence. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to the TRAC gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is up to 15 nucleotides, up to 20 nucleotides, up to 25 nucleotides, up to 30 nucleotides, up to 35 nucleotides, up to 40 nucleotides, up to 55 nucleotides, up to 60 nucleotides, up to 65 nucleotides, up to 70 nucleotides, up to 75 nucleotides, up to 80 nucleotides, up to 85 nucleotides, up to 90 nucleotides, up to 95 nucleotides, or up to 100 nucleotides in length. In certain embodiments, the RNAi agent comprises DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0455] In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of TCR by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% or any intermediate value or range thereof. In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of TCR by about 20%. In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of TCR by about 30%.

[0456] In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene disruption of a TRBC locus (e.g., a TRBC1 locus, a TRBC2 locus). In certain embodiments, the gene disruption of the TRBC locus (e.g., a TRBC1 locus, a TRBC2 locus) results in a non-functional TCR. In certain embodiments, the gene disruption of the TRBC locus (e.g., a TRBC1 locus, a TRBC2 locus) results in knockout of the TCR gene expression.

[0457] Any methods to generate the gene disruption of the CD70 locus, as disclosed above, can be used to generate the gene disruption of the TRBC locus (e.g., a TRBC1 locus, a TRBC2 locus). In certain embodiments, the gene disruption of the TRBC locus is generated by a method comprising a gene editing method comprising homologous recombination, a Zinc finger nuclease, a meganuclease, a Transcription activator-like effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0458] In certain embodiments, the gene disruption of the TRBC locus can be a disruption of the coding region of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption of the coding region of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion at the coding region of the TRBC1 locus. Human TRBC1 protein comprises 4 exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRBC1 locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption at one or more of exon 1 through exon 4 of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption at exon 1 of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion at exon 1 of the TRBC1 locus.

[0459] In certain embodiments, the gene disruption of the TRBC locus can be a disruption of the coding region of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption of the coding region of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion at the coding region of the TRBC2 locus. Human TRBC2 protein comprises 4 exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRBC2 locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption at one or more of exon 1 through exon 4 of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption at exon 1 of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion at exon 1 of the TRBC2 locus.

[0460] In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene modification of a TRBC gene. The gene modification of the TRBC gene can result in a non-functional TCR protein or a knockdown of the TCR gene expression. In certain embodiments, the gene modification of the TRBC gene results in knockout of the TCR gene expression.

[0461] In certain embodiments, the modification of the TRBC gene comprises use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent comprises a shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the TRBC gene and thereby reduces or eliminates the expression of the TRBC gene or TCR protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses the first antigen-recognizing receptor and / or the second antigen-recognizing receptor disclosed herein. In certain embodiments, the expressions of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor are driven by identical promoters (e.g., a same promoter). In certain embodiments, the expressions of the shRNA, the first antigen-recognizing receptor, and the second antigen-recognizing receptor disclosed herein are driven by different promoters.

[0462] In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, 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 about 100% homologous or identical to at least a portion of a TRBC nucleic acid sequence. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to the TRBC gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is up to 15 nucleotides, up to 20 nucleotides, up to 25 nucleotides, up to 30 nucleotides, up to 35 nucleotides, up to 40 nucleotides, up to 55 nucleotides, up to 60 nucleotides, up to 65 nucleotides, up to 70 nucleotides, up to 75 nucleotides, up to 80 nucleotides, up to 85 nucleotides, up to 90 nucleotides, up to 95 nucleotides, or up to 100 nucleotides in length. In certain embodiments, the RNAi agent comprises DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0463] In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of TCR by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% or any intermediate value or range thereof. In certain embodiments, the RNAi agent reduces the expression (e.g., endogenous expression) of TCR by about 60%.

[0464] The presently disclosed cells can be isolated and activated by using CD3 / CD28 antibodies before the generation of a gene disruption. In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene disruption of a TRAC locus, a TRBC locus, and / or of a CD70 locus. In certain embodiments, a presently disclosed cell comprising an antigen-recognizing receptor comprising a pTCR polypeptide further comprises a gene modification of a TRAC locus, a TRBC locus, and / or of a CD70 locus.

[0465] In certain embodiments, the gene disruption of the TRAC locus, the gene disruption of the TRBC locus, and / or the gene disruption of the CD70 locus are generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene disruption of the TRAC locus, the gene disruption of the TRBC locus, and / or the gene disruption of the CD70 locus are generated before isolation and activation of the cells (e.g., T cells).

[0466] In certain embodiments, the gene disruption of the TRAC locus and / or TRBC locus is generated before isolation and activation of the cells (e.g., T cells) and the gene disruption of the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene disruption of the CD70 locus is generated before isolation and activation of the cells (e.g., T cells) and the gene disruption of the TRAC locus and / or TRBC locus is generated after isolation and activation of the cells (e.g., T cells).

[0467] In certain embodiments, the gene modification of the TRAC locus, the gene modification of the TRBC locus, and / or the gene modification of the CD70 locus are generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene modification of the TRAC locus, the gene modification of the TRBC locus, and / or the gene modification of the CD70 locus are generated before isolation and activation of the cells (e.g., T cells).

[0468] In certain embodiments, the gene modification of the TRAC locus and / or TRBC locus is generated before isolation and activation of the cells (e.g., T cells) and the gene modification of the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene modification of the CD70 locus is generated before isolation and activation of the cells (e.g., T cells) and the gene modification of the TRAC locus and / or TRBC locus is generated after isolation and activation of the cells (e.g., T cells).

[0469] 5. Formulations and Administration The presently disclosed subject matter provides compositions comprising presently disclosed cells (e.g., disclosed in Section 4). In certain embodiments, the compositions are pharmaceutical compositions that further comprise a pharmaceutically acceptable excipient.

[0470] Compositions comprising the presently disclosed cells can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, disper...

Claims

WHAT IS CLAIMED IS:

1. An antigen-recognizing receptor comprising an extracellular antigen-binding domain that binds to an antigen, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises a pre-T cell receptor (pTCR) polypeptide.

2. The antigen-recognizing receptor of claim 1, wherein the pTCR polypeptide comprises a pTCRa polypeptide or a fragment thereof.

3. The antigen-recognizing receptor of claim 1, wherein the pTCR polypeptide comprises an intracellular domain of a pTCRa polypeptide or a fragment thereof.

4. The antigen-recognizing receptor of claim 1, wherein the pTCR polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 17.

5. The antigen-recognizing receptor of claim 4, wherein the pTCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17.

6. The antigen-recognizing receptor of claim 1, wherein the pTCR polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 18.

7. The antigen-recognizing receptor of claim 6, wherein the pTCR polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18.

8. The antigen-recognizing receptor of claim 1, wherein the antigen is a tumor antigen or a pathogen antigen.

9. The antigen-recognizing receptor of claim 8, wherein the antigen is a tumor antigen.

10. The antigen-recognizing receptor of claim 8, wherein the tumor antigen is selected from CD 19, carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD8, CD7, CD 10, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein 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 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-ESO-1,oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, PRAME CCR4, CD5, CD3, TRBC1, TRBC2, TIM-3, Integrin B7, ICAM-1, CD70, Tim3, CLEC12A, and ERBB.

11. The antigen-recognizing receptor of claim 8, wherein the tumor antigen is CD 19 or CD70.

12. The antigen-recognizing receptor of claim 1, wherein the antigen-recognizing receptor is a chimeric antigen receptor (CAR), a chimeric costimulatory receptor (CCR), or a HLA-independent T cell receptor (HIT).

13. The antigen-recognizing receptor of claim 12, wherein the antigen-recognizing receptor is a chimeric antigen receptor (CAR).

14. The antigen-recognizing receptor of claim 13, wherein the intracellular signaling domain of the CAR comprises a CD3(^ polypeptide.

15. The antigen-recognizing receptor of claim 14, wherein the CD3(^ polypeptide is a native CD3(^ polypeptide or a modified CD3(^ polypeptide.

16. The antigen-recognizing receptor of claim 15, wherein the native CD3(^ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 21.

17. The antigen-recognizing receptor of claim 15, wherein the modified CD3(^ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations.

18. The antigen-recognizing receptor of claim 17, wherein the modified CD3(^ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 34.

19. The antigen-recognizing receptor of claim 13, wherein the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region.

20. The antigen-recognizing receptor of claim 19, wherein the at least one costimulatory signaling region comprises at least an intracellular domain of a co-stimulatory molecule or a portion thereof.

21. The antigen-recognizing receptor of claim 20, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, CD27, CD40, CD154, CD97, CDlla / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

22. The antigen-recognizing receptor of claim 21, wherein the costimulatory molecule comprises a CD28 polypeptide.

23. The antigen-recognizing receptor of claim 21, wherein the costimulatory molecule comprises amino acids 180 to 220 of SEQ ID NO: 15.

24. The antigen-recognizing receptor of claim 13, wherein the CAR comprises a transmembrane domain.

25. The antigen-recognizing receptor of claim 12, wherein the antigen-recognizing receptor is a chimeric costimulatory receptor (CCR).

26. The antigen-recognizing receptor of claim 25, wherein the intracellular domain of the CCR does not alone deliver an activation signal to the cell.

27. The antigen-recognizing receptor of claim 25, wherein the intracellular domain of the CCR further comprises at least an intracellular domain of a co-stimulatory molecule or a portion thereof.

28. The antigen-recognizing receptor of claim 27, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, CD27, CD40, CD154, CD97, CDlla / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

29. The antigen-recognizing receptor of claim 12, wherein the antigen-recognizing receptor is HLA-independent T cell receptor (HIT).

30. The antigen-recognizing receptor of claim 29, wherein the HIT 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 antigen-binding fragment of a light chain variable region (VL) of the antibody; wherein each of the first and second antigen-binding chains comprises the TRAC polypeptide or the TRBC polypeptide, and wherein the HIT binds to the antigen in an HLA-independent manner.

31. The antigen-recognizing receptor of claim 30, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous.

32. The antigen-recognizing receptor of claim 30, wherein the first and the second antigenbinding chains bind to the antigen with a dissociation constant (KD) of about 1 x 10'8M or less.

33. The antigen-recognizing receptor of claim 30, wherein the first and the second antigenbinding chains bind to the antigen with a dissociation constant (KD) of about 5 x 10'9M or less.

34. The antigen-recognizing receptor of claim 30, wherein the first antigen-binding chain comprises an antigen-binding fragment of a VH of an antibody and a TRBC polypeptide, and the second antigen-binding chain comprises an antigen-binding fragment of a VL of the antibody and a TRAC polypeptide.

35. The antigen-recognizing receptor of claim 30, wherein the first antigen-binding chain comprises an antigen-binding fragment of a VH of an antibody and a TRAC polypeptide, and the second antigen-binding chain comprises an antigen-binding fragment of a VL of the antibody and a TRBC polypeptide.

36. The antigen-recognizing receptor of claim 30, wherein the first and second antigen binding chains are capable of associating with a CD3(^ polypeptide.

37. The antigen-recognizing receptor of claim 30, wherein the first and second antigen binding chains, upon binding to the second antigen, are capable of activating the CD3(^ polypeptide.

38. A nucleic acid encoding an antigen-recognizing receptor of any one of claims 1-37.

39. The nucleic acid of claim 38, further comprising a promoter that is operably linked to the antigen-recognizing receptor.

40. The nucleic acid of claim 38, wherein the promoter is selected from the group consisting of an elongation factor (EF)-l promoter, a CMV promoter, a SV40 promoter, a PGK promoter, and a metallothionein promoter.

41. The nucleic acid of claim 38, wherein the promoter is an inducible promoter selected from the group consisting of a NF AT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, and an IL-2 promoter.

42. A vector comprising the nucleic acid of any one of claims 38-41.

43. A lipid nanoparticle comprising the nucleic acid of any one of claims 38-41.

44. A cell comprising an antigen-recognizing receptor of any one of claims 1-37, the nucleic acid of any one of claims 38-41, the vector of claim 42, or the lipid nanoparticle of claim 43.

45. The cell of claim 44, wherein the antigen-recognizing receptor is recombinantly expressed.

46. The cell of claim 44 or 45, wherein the antigen-recognizing receptor is expressed from a vector.

47. The cell of any one of claims 44-46, wherein the cell is a cell of the lymphoid lineage or a cell of the myeloid lineage.

48. The cell of claim 47, wherein the cell of the lymphoid lineage is selected from T cells, B cells, Natural Killer (NK) cells, and dendritic cells.

49. The cell of any one of claims 44-47, wherein the cell is a T cell.

50. The cell of claim 49, wherein the T cell is derived from an induced pluripotent stem cell.

51. The cell of claim 49 or 50, wherein the T cell is a CD8+T cell.

52. The cell of claim 51, wherein the CD8+T cell is CD4 independent.

53. The cell of any one of claims 48-57, wherein the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a yb T cell, a tumor-infiltrating lymphocyte (TIL), a virus-specific T cell (VST), a regulatory T cell, and a Natural Killer T (NKT) cell.

54. The cell of any one of claims 48-53, wherein the T cell is CD62L+.

55. The cell of any one of claims 48-53, wherein the T cell is CD45RA+.

56. The cell of any one of claims 48-53, wherein the T cell is CD45RA+and CD62L+.

57. The cell of claim 53, wherein the T cell is a tumor-infiltrating lymphocyte (TIL).

58. The cell of claim 53, wherein the T cell is a virus-specific T cell (VST).

59. The cell of any one of claims 44-48, wherein the cell is an NK cell.

60. The cell of any one of claims 44-58, wherein the antigen-recognizing receptor is integrated at a locus within the genome of the T cell.

61. The cell of claim 60, wherein the locus is selected from the group consisting of a TRAC locus, a TRBC locus, a TRDC locus, and a TRGC locus.

62. The cell of claim 60 or 61, wherein the locus is a TRAC locus or a TRBC locus.

63. The cell of any one of claims 60-62, wherein the locus is a TRAC locus.

64. The cell of any one of claims 44-63, wherein the expression of the antigen-recognizing receptor is under the control of an endogenous promoter.

65. The cell of claim 64, wherein the endogenous promoter is selected from the group consisting of an endogenous TRAC promoter, an endogenous TRBC promoter, an endogenous TRDC promoter, an endogenous TRGC promoter, and a combination thereof.

66. The cell of claim 64 or 65, wherein the endogenous promoter is a TRAC promoter.

67. The cell of any one of claims 44-66, wherein the cell further comprises a gene disruption of a TRAC locus and / or a TRBC locus.

68. The cell of any one of claims 44-67, wherein the cell further comprises a gene disruption of a CD70 locus.

69. The cell of any one of claims 44-68, wherein the cell further comprises a gene disruption of a TRAC locus, a TRBC locus, and / or a CD70 locus.

70. The cell of any one of claims 44-69, wherein the cell further comprises a gene modification of a TRAC gene and / or a TRBC gene.

71. The cell of any one of claims 44-70, wherein the cell further comprises at least one exogenous costimulatory ligand.

72. The cell of claim 71, wherein the at least one exogenous co-stimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof.

73. The cell of claim 72, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), CD40L.

74. The cell of claim 72, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

75. The cell of any one of claims 71-74, wherein the at least one exogenous costimulatory ligand comprises CD80.

76. The cell of any one of claims 71-75, wherein the at least one exogenous a costimulatory ligand comprises 4-1BBL.

77. The cell of any one of claims 71-76, wherein the cell comprises two exogenous costimulatory ligands.

78. The cell of any one of claims 71-77, wherein the at least two exogenous costimulatory ligands comprise CD80 and 4-1BBL.

79. The cell of claim 77 or 78, wherein the at least two exogenous costimulatory ligands comprise the amino acid sequence set forth in SEQ ID NO: 67 and / or the amino acid sequence set forth in SEQ ID NO: 69.

80. The cell of any one of claims 44-79, wherein the cell further comprises a fusion polypeptide comprising: a) an extracellular domain and a transmembrane domain of a costimulatory ligand, and b) an intracellular domain of a first co-stimulatory molecule.

81. The cell of claim 80, wherein the co-stimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and combinations thereof.

82. The cell of claim 81, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof.

83. The cell of claim 81, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

84. The cell of any one of claims 80-83, wherein the co-stimulatory ligand is CD80.

85. The cell of any one of claims 80-84, wherein the first co-stimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

86. The cell of any one of claims 80-85, wherein the first co-stimulatory molecule is 4-1BB.

87. The cell of any one of claims 80-86, wherein the co-stimulatory ligand is CD80 and the first co-stimulatory molecule is 4-1BB.

88. The cell of any one of claims 80-87, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 71.

89. The cell of any one of claims 80-88, wherein the fusion polypeptide further comprises an intracellular domain of a second co-stimulatory molecule.

90. The cell of claim 89, wherein the second co-stimulatory molecule is selected from the group consisting of CD28, 4-1BB, 0X40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

91. The cell of claim 89 or 90, wherein the second co- stimulatory molecule is CD28.

92. The cell of any one of claims 89-91, wherein the co-stimulatory ligand is CD80, the first co-stimulatory molecule is 4-1BB, and the second co-stimulatory molecule is CD28.

93. The cell of any one of claims 89-92, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 72.

94. A cell comprising (a) a first antigen-recognizing receptor comprising a pre-T cell receptor (pTCR) polypeptide, and (b) a second antigen-recognizing receptor.

95. The cell of claim 94, wherein(a) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a CAR;(b) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a CCR;(c) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a HIT;(d) the first antigen-recognizing receptor is a CAR and the second antigen-recognizing receptor is a recombinant TCR;(e) the first antigen-recognizing receptor is a CCR and the second antigen-recognizing receptor is a CAR;(f) the first antigen-recognizing receptor is a CCR and the second antigen-recognizing receptor is a HIT; or(g) the first antigen-recognizing receptor is a CCR and the second antigen-recognizing receptor is a recombinant TCR.

96. The cell of any one of claims 44-95, wherein said cell is autologous.

97. The cell of any one of claims 44-95, wherein said cell is allogeneic.

98. A composition comprising the cell of any one of claims 44-97, the nucleic acid of any one of claims of any one of claims 38-41, the vector of claim 42, or the lipid nanoparticle of claim 43.

99. The composition of claim 98, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

100. A method for producing a modified cell, the method comprising introducing into the cell the nucleic acid of any one of claims of any one of claims 38-41, the vector of claim 42, the lipid nanoparticle of claim 43, or the composition of claim 98 or 99.

101. A cell produced by the method of claim 100.

102. A method of reducing tumor burden in a subject, the method comprising administering to the subject an effective amount of the cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99.

103. The method of claim 102, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

104. A method of preventing and / or treating a neoplasm or a tumor in the subject, administering to the subject an effective amount of the cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99.

105. The method of any one of claims 102-104, wherein the neoplasm or tumor is cancer.

106. The method of any one of claims 102-105, wherein the neoplasm or tumor comprises antigen heterogeneity of the antigen.

107. The method of claim 106, wherein the antigen has a low antigen density.

108. The method of claim 106 or 107, wherein the antigen is expressed on tumor cells having a low tumor cell frequency.

109. The method of any one of claims 102-108, wherein the neoplasm or tumor is a solid tumor.

110. The method of claim 110, wherein the solid tumor is selected from the group consisting of melanoma, renal cell carcinoma, non-small-cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung neuroendocrine carcinoma, small-cell lung cancer, pancreatic cancer, breast cancer, astrocytoma, glioblastoma, laryngeal / pharyngeal carcinoma, EBV-associated nasopharyngeal carcinoma, and ovarian carcinoma.

111. The method of any one of claims 102-108, wherein the neoplasm or tumor is a blood cancer.

112. The method of claim 111, wherein the neoplasm or tumor is a myeloid disorder.

113. The method of claim 112, wherein the myeloid disorder is selected from the group consisting of myelodysplastic syndromes, myeloproliferative neoplasms, chronic myelomonocytic leukemia, or acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelocytic leukemia, and polycythemia vera.

114. The method of claim 111 or 112, wherein the myeloid disorder is acute myeloid leukemia (AML).

115. The method of claim 111, wherein the neoplasm or tumor is a B-cell malignancy.

116. The method of claim 115, wherein the B-cell malignancy is selected from the group consisting of B cell non-Hodgkin lymphoma (NHL), B cell Hodgkin's lymphoma, B cell acutelymphocytic leukemia (ALL), B cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter’s transformation, and CNS lymphoma.

117. The method of claim 115 or 116, wherein the B-cell malignancy is B cell acute lymphocytic leukemia.

118. The method of claim 111, wherein the neoplasm or tumor is a leukemia.

119. The method of claim 118, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed-phenotype acute leukemia (MLL), hairy cell leukemia, B cell prolymphocytic leukemia, B-cell precursor acute lymphoblastic leukemia, and T-cell precursor acute lymphoblastic leukemia.

120. The method of claim 111, wherein the neoplasm or tumor is a lymphoma.

121. The method of claim 120, wherein the lymphoma is selected from the group consisting of Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, B-cell non-Hodgkin’s lymphoma, T-cell non-Hodgkin’s lymphoma, and T-cell precursor acute lymphoblastic lymphoma.

122. The method of any one of claims 102-121, wherein the subject has a relapse of the neoplasm or tumor.

123. The method of any one of claims 102-122, wherein the subject received treatment which leads to residual tumor cells.

124. A method of preventing and / or treating a pathogen infection in a subject, the method comprising administering to the subject an effective amount of the cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99.

125. A method of preventing and / or treating an autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99.

126. A method of preventing and / or treating an infectious disease in a subject, the method comprising administering to the subject an effective amount of the cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99.

127. The cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99 for use in reducing tumor burden, treating and / or preventing a neoplasm or a tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease in a subject.

128. Use of the cells of any one of claims 44-97 or 101 for the manufacturing of a medicament for reducing tumor burden, treating and / or preventing a neoplasm or a tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease in a subject.

129. A kit comprising the cells of any one of claims 44-97 or 101, or the composition of claim 98 or 99.

130. The kit of claim 129, wherein the kit further comprises written instructions for reducing tumor burden, treating and / or preventing a neoplasm or a tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease.

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