Compositions and methods for modulating TCR specificity

Abstract

The present technology provides compositions and methods for modulating T Cell Receptor (TCR) specificity as well as methods for treating cancer in a subject in need thereof. The present disclosure provides engineered cytotoxic T cells comprising a TCR and / or nucleic acid encoding the TCR and a mutant CD8 alpha polypeptide and / or nucleic acid encoding the mutant CD8 alpha polypeptide.

Description

Atty. Dkt. No.: 115872-3443COMPOSITIONS AND METHODS FOR MODULATING TCR SPECIFICITY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 760,518, filed February 19, 2025, and U. S. Provisional Patent Application No. 63 / 916,280, filed November 12, 2025, the contents of which are incorporated herein by reference in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA241894 awarded by the National Institutes of Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present technology relates generally to compositions and methods for modulating T Cell Receptor (TCR) specificity as well as methods for treating cancer in a subject in need thereof. The present disclosure provides engineered cytotoxic T cells comprising a TCR and / or nucleic acid encoding the TCR and a mutant CD8 alpha polypeptide and / or nucleic acid encoding the mutant CD8 alpha polypeptide.BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] Adoptive cell therapies, in which T cells are exogenously engineered to express a chimeric antigen receptors (CAR), T cell receptors (TCR), or TCR-mimic (TCRm) single chain fragment variable (scFv), that redirect them to tumor associated antigens have been effective as cancer treatments. TCRs and TCRms recognize peptides presented on the cell surface in the context of major histocompatibility complexes (MHC). These peptides are derived from proteins from any cellular location and are therefore not limited to dysregulated, overexpressed, or lineage-specific proteins found on the cell surface, as is the case with CAR T cells and traditional antibodies, thus vastly expanding the repertoire of potentially targetable cancer antigens.

[0006] TCRs recognize amino acids as short linear peptides, typically 8-12 amino acids long for MHC class I (MHCI) restricted TCRs, buried in the groove of an MHC protein. Hence, cross-reactivities with presented peptides that have similar amino acid sequences are frequently observed. A major drawback of TCR-based therapies is that it is extremely challenging to-1- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443predict off-target reactivities that can lead to toxicides or to modulate these detrimental cross reactions. This problem was made vividly evident by severe toxicides associated with some TCR therapies, for example the MAGE-A3 TCR T cells, which resulted in fatal cardiotoxicity due to unpredicted cross reactivity with a peptide derived from cardiac muscle titin protein.

[0007] Accordingly, there remains an urgent need for efficient methods to predict and prevent off-target reactivities and potential toxicides of highly promiscuous TCR-based agents. In addition, methods to mitigate the cross-reactions do not currently exist, even if identified.SUMMARY OF THE PRESENT TECHNOLOGY

[0008] In one aspect, the present disclosure provides an engineered cytotoxic T cell that comprises (a) a T cell receptor (TCR) that binds to a target antigen and / or a nucleic acid encoding the T cell receptor; (b) lacks detectable expression or activity of a wild-type CD8 alpha polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; and (c) comprises a non-endogenous expression vector that includes a nucleic acid sequence encoding a mutant CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62, optionally wherein the nucleic acid sequence of the mutant CD8 alpha polypeptide is operably linked to an expression control sequence. Examples of expression control sequences include, but are not limited to, inducible promoters, constitutive promoters, native promoters, or heterologous promoters.

[0009] In some embodiments of the engineered cytotoxic T cell of the present disclosure, the TCR is a native TCR, a non-native TCR, or a mimic TCR. Examples of mimic TCRs include but are not limited to, TCRm Abs, TCRm BITEs, TCRm CARs, and ImmTACs. Additionally or alternatively, in some embodiments, the TCR is IG4, DMF5, TCR4, MAGE A3, or A6. In certain embodiments, the engineered cytotoxic T cell of the present disclosure is derived from an autologous donor or an allogeneic donor.

[0010] Additionally or alternatively, in some embodiments of the engineered cytotoxic T cell disclosed herein, the mutant CD8 alpha polypeptide exhibits reduced binding to major histocompatibility complex (MHC) relative to the wild-type CD8 alpha polypeptide.

[0011] Additionally or alternatively, in some embodiments, the engineered cytotoxic T cell comprises a deletion, an inversion, a missense mutation, a nonsense mutation, or a frameshift mutation in a nucleic acid sequence encoding the wild-type CD8 alpha polypeptide, optionally wherein the nucleic acid sequence encoding the wild-type CD8 alpha polypeptide is SEQ ID NO: 1. In certain embodiments, the engineered cytotoxic T cell comprises an inhibitory nucleic acid that specifically targets and inhibits the expression of a nucleic acid sequence encoding the wild-type CD8 alpha polypeptide, optionally wherein the nucleic acid sequence -2- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443encoding the wild-type CD8 alpha polypeptide is SEQ ID NO: 1. In some embodiments, the inhibitory nucleic acid is an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA.

[0012] In another aspect, the present disclosure provides an engineered CD4+ helper T cell that comprises (a) a T cell receptor that binds to a target antigen and / or a nucleic acid encoding the T cell receptor; and (b) comprises a non-endogenous expression vector that includes a nucleic acid sequence encoding a CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62, and / or is operably linked to an expression control sequence. Examples of expression control sequences include, but are not limited to, inducible promoters, constitutive promoters, native promoters, or heterologous promoters.

[0013] In some embodiments of the engineered CD4+ helper T cell disclosed herein, the TCR is a native TCR, a non-native TCR, or a mimic TCR. Examples of mimic TCRs include but are not limited to, TCRm Abs, TCRm BITEs, TCRm CARs, and ImmTACs. In certain embodiments, the TCR is IG4, DMF5, TCR4, MAGE A3, or A6. In certain embodiments, the engineered CD4+ helper T cell is derived from an autologous donor or an allogeneic donor.

[0014] Additionally or alternatively, in some embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell described herein, the non-endogenous expression vector is a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector.

[0015] In any and all embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell disclosed herein, the target antigen comprises a tumor antigen. Examples of suitable tumor antigens include, but are not limited to, Tyrosinase, NY-ESO-1, CD277-mediated presentation, MAGE-A4, WT1, MAGE-A10, PRAME, EBV LMP2, MAGE-A1, HA-1, HERV-E, CMV pp65, HBV, TRAIL-DR4, HIV SL9, and AFP.

[0016] In one aspect, the present disclosure provides a composition comprising an effective amount of any and all embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell described herein, and a pharmaceutically acceptable carrier.

[0017] In another aspect, the present disclosure provides a kit comprising an expression vector that includes a nucleic acid sequence encoding a CD8 alpha amino acid sequence of one or more of any one of SEQ ID NOs: 4-53 or 57-62, and instructions for transducing CD4+ helper T cells with the expression vector. The kit may further comprise a vector encoding an engineered T-cell receptor (TCR) that binds to a target antigen.

[0018] In yet another aspect, the present disclosure provides a kit comprising an expression vector that includes a nucleic acid sequence encoding a mutant CD8 alpha amino acid sequence of one or more of any one of SEQ ID NOs: 4-53 or 57-62, and instructions for -3- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443transducing cytotoxic T cells with the expression vector. The kit may further comprise a vector encoding an engineered T-cell receptor (TCR) that binds to a target antigen.

[0019] Also disclosed herein are methods for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of any and all embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell described herein, or a composition comprising an effective amount of any and all embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell described herein, and a pharmaceutically acceptable carrier.

[0020] In one aspect, the present disclosure provides a method for mitigating off-target reactivity / toxicity in a subject receiving adoptive T cell therapy comprising administering to the subject an effective amount of any and all embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell described herein, or a composition comprising an effective amount of any and all embodiments of the engineered cytotoxic T cell or the engineered CD4+ helper T cell described herein, and a pharmaceutically acceptable carrier.

[0021] In any of the preceding embodiments of the methods disclosed herein, the subject suffers from or is diagnosed with cancer. In certain embodiments, the cancer or tumor is selected from the group consisting of adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.

[0022] Additionally or alternatively, in some embodiments of the methods disclosed herein, the engineered cytotoxic T cell or engineered CD4+ helper T cell is administered pleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.

[0023] In another aspect, the present disclosure provides, among other things, a method of preparing cytotoxic T cells for adoptive cell therapy comprising: isolating cytotoxic T cells from a donor subject; inactivating expression and / or activity of a wild-type CD8 alpha polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 in the -4- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443cytotoxic T cells; transducing the cytotoxic T cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a mutant CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62; and administering the transduced cytotoxic T cells to a recipient subject. In yet another aspect, the present disclosure provides a method of preparing CD4+ helper T cells for adoptive cell therapy comprising: isolating CD4+ helper T cells from a donor subject; transducing the CD4+ helper T cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62; and administering the transduced CD4+ helper T cells to a recipient subject. In some embodiments, a donor subject and a recipient subject are the same or different. In some embodiments of the methods disclosed herein, the transduced cytotoxic T cells or the transduced CD4+ helper T cells comprise a native T cell receptor (TCR), a non-native TCR, or a mimic TCR. Examples of mimic TCRs include but are not limited to, TCRm Abs, TCRm BITEs, TCRm CARs, and ImmTACs.BRIEF DESCRIPTION OF THE DRAWINGS|'<)024] FIG. 1: Schematic overview of CD8 stabilizing the pMHC complex. CD8 is a glycoprotein expressed on the surface of CD8+ T cells. It consists of two chains, CD8a and CD8p. The binding of CD8 to MHCI is primarily mediated through the CD8a chain's immunoglobulin-like domain, which interacts with the a3 domain of the MHCI. This interaction is non-covalent and involves several non-covalent forces, including hydrogen bonds, hydrophobic interactions, and salt bridges. Intracellular p56lckcan bind to CD8a.

[0025] FIGs. 2A-2F: CD8aa Reduces Off-Target Reactivity While Preserving Cytotoxic Function (1G4 LY is affinity enhanced TCR; 1G4 TS is native TCR). FIG. 2A: Flow plot showing TCR expression, CD8A and CD8B expression of CD8, CD8aa, and CD8KO 1G4 T cells. FIG. 2B: IFN-y ELISPOT comparing CD8, CD4, CD8aa, and CD8 KO T cells with 1G4 LY, showing reduced off-target reactivity in CD8aa. FIGs. 2B-2C: Cytotoxicity of 1G4 LY T cells with CD8, CD8aa, and CD8KO against A375 and SKMEL37 melanoma cells across E: T ratios, with CD8aa retaining killing ability. FIGs. 2D-2F: Killing assay of 1G4 TS variant with and without additional CD8a, indicating that CD8a addition alone does not enhance 1G4 TS cytotoxicity.

[0026] FIGs. 3A-3E: Strategic Identification of CD8a Variants that Enhance TCR Signaling for On-Target Specificity Using Jurkat-NFAT-eGFP Reporter System: serially enriching mutations in CD8. FIG. 3A: Schematic overview of the pipeline for saturation mutagenesis screening of CD8a mutants. JurkatNFAT-eGFP reporter cells were co-cultured -5- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443with T2 cells pulsed with NY-ESO-1 peptide (0.2 pg / mL) or no peptide (control). Cells were sorted for the top 5% GFP+ cells after 24 hours, sequenced, and normalized to the control. Hits were validated in live-cell cancer killing assays. FIG. 3B: Structural model (PDB: 1AKJ) with CD8a CDR regions (red) near MHC class I (wheat) and P2m (yellow) [Leishman et al., J Exp Med, 1991], FIG. 3C: Schematic overview of the enrichment screen process. After sorting the top 5% GFP+ cells, cells were re-cultured for three additional rounds. DNA was extracted from harvested cells after each round, and amplicon sequencing of CD8a revealed enriched mutants across the screening rounds. FIG. 3D: Heatmap showing the log2 fold changes (log2FC) of CD8a variants across three independent screens. The Y-axis shows represents CD8a variants, with highly enriched variants colored in red while low enriched or depleted variants are shown in blue. FIG. 3E: Top 25 CD8a variants, with fold-change data across rounds in three replicates.

[0027] FIGs. 4A-4C: Sequence diversity and functional impact of CD8 mutants. FIG. 4A: Alignment of full-length HLA amino acid sequences, highlighting conserved and polymorphic regions [PD-IMGT / HLA Database, www.ebi.ac.uk / ipd / imgt / hla], FIG. 4B: Number of amino acid differences per HLA subdomain. FIG. 4C: Different HLA and TCR combinations will be tested.

[0028] FIGs. 5A-5F: Validation of Hits using Flow Cytometry and Cytotoxicity Assays across two TCR / HLA pairs. FIGs. 5A-5B: Flow cytometry analysis of CD8a mutants in combination with 1G4 TCR and TCR4 reactive against the PIK3CA neoantigen presented in HLA*A03 context [Chandran et al., Nat Med, 2022], CD8beta expression significantly increases in specific CD8a mutants. FIG. 5C: Cytotoxicity of CD8a mutants with native 1G4 TCR against A02+ A375 cell line. FIG. 5D: Cytotoxicity of CD8a mutants with native TCR4 against A03+ HCC70 / PIK3CAmut cell line. FIG. 5E: Cytokine expression in T cells expressing the top CD8a mutant hits and TCR4 and control cells (TCR4 CD8aP) following stimulation with mut PIK3CA peptide pulsed COS7 / A03+ cells for 5 hours. Cells were gated on TCR4 expression, followed by ICS to assess IFNy, IL-2, and TNFa production. FIG. 5F:Individual IncuCyte plots of the top-performing CD8a mutants, showing the most effective variants.

[0029] FIG. 6: CD8 variants maintain off-target reactivity of native TCR. IFN-y ELISPOT comparing leading mutants for off-target reactivity. Mutants do not enhance responses to previously identified off-targets reactive with affinity enhanced 1G4 TCR (iron).

[0030] FIG. 7: Validation of CD8 mutants in native CD8«p 1G4 TS cells. CD8 T cells expressing CD8 variants and the 1G4 TCR were evaluated for cytotoxic activity using an -6- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443IncuCyte live-cell imaging assay. T cells were co-cultured with A375 target cells expressing NLS-mCherry at an effector-to-target (E: T) ratio of 4: 1 for 96 hours. Red Fluorescent Units (RCU / μm2per image), normalized to t=0, were plotted over time to assess target cell survival. Some CD8 variants enhanced cytotoxicity compared to the native TCR, TCR+CD8a WT, and TCR+CD8aP WT. Data represent the average of three donors.

[0031] FIG. 8A: CD8a Variant Testing with TCR4 Against PIK3CA Neoepitope. CD8a variants were tested in combination with TCR4 for recognition of the PIK3CA neoepitope. Flow cytometry analysis revealed that some CD8a variants resulted in increased CD8a and CD8P surface expression. FIG. 8B: Functional Avidity of CD8a Variants. Intracellular cytokine staining was performed to assess the functional avidity of all CD8a variants. T cells were co-cultured with COS7 cells transduced with HLA-A*03 and pulsed with increasing concentrations of the PIK3CA neoepitope (0, 0.002, 0.02, 0.2, 2, and 20 pg / mL). IFNy, IL2, and CD 107a expression were measured by flow cytometry. Some CD8a variants exhibited higher cytokine expression compared to control cells expressing TCR4 alone, TCR4+CD8a WT, and TCR4+CD8aP WT. Representative flow cytometry plots are shown for select mutants at a peptide concentration of 2 pg / mL.

[0032] FIG. 9: Summary of T cell types generated.

[0033] FIG. 10: Enhanced functional avidity for some CD8a mutants against PIK3CA mutant peptides.

[0034] FIG. 11: Correlation between CD8 stability and cytotoxicity.

[0035] FIG. 12: CD8a mutants significantly reduce CD8|J expression.

[0036] FIGs. 13A-13F: CD8a saturation mutagenesis screen to enhance TCR functionality. FIG. 13A: Schematic representation of the plasmid constructs used to express TCRa and TCRP sequences, separated by a 2A peptide sequence, followed by CD8a or CD8a and CD8P (also separated by a 2A peptide). FIG. 13B: Representative cancer killing curve of the A375 cancer cell line expressing mCherry NLS, co-cultured with T cells expressing either affinity-enhanced 1G4 / LY, native 1G4 / TS, native 1G4 / TS + CD8a, 1G4 / TS + CD8aP, or untransduced (UT) T cells. Killing is measured by Total Red Integrated Intensity per area, normalized to t=0 for each well. Data are shown as mean ± SEM. FIG. 13C: Structural diagram showing the locations of mutated residues in a subunit of the CD8a CDRregions (L46-S52, S74-N76, N120-S121) that were altered to all other amino acids (PDB: 1 AKJ) in the library. These regions are involved in pMHC binding and TCR signaling. The screen included 228 mutant variants. FIG. 13D: Flow cytometry plots showing GFP expression of the Jurkat NF AT GFP+ CD8a variant library. Left panel: resting library cells.-7- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443Right panel: library cells co-cultured with 0.2 ug / ml NY-ESO-1 peptide for 24 hours. FIG. 13E: Flow cytometry data showing the percentage of GFP+ cells (TCR-activated) after each of the three rounds of sorting for one representative screen, indicating consistentenrichment of TCR-activated cells when co-cultured with NY-ESO-1-pulsed T2 cells. FIG. 13F: Volcano plot showing the log2 fold change (log2FC) of enriched CD8a mutations after three rounds of screening, with 17 mutations demonstrating significant enrichment (log2FC >2, p<0.05), highlighting promising candidates for further functional validation.

[0037] FIGs. 14A-14H: Top CD8 mutants enhance TCR cytotoxicity across multiple HLA / TCR contexts. FIGs. 14A-14D: Live-cell imaging of A375 melanoma cells (NY-ESO-1+, HLA-A*02:01+) co-cultured with T cells expressing 1G4 / TS TCR or 1G4 / TS and mutant CD8 (L46S, L46T, L47Q, and S121N). Tumor cell killing was evaluated across multiple donors (full data set is shown in Extended data Fig. 3b-g). Raw confluence values were exported and analyzed in R (v4.3.3). Each condition included three technical replicates per donor. Data were analyzed by unpaired two-sample t-tests (Welch’s correction) comparing each condition to the control (1G4 / TS + CD8aP or 1G4 / TS). Tests were performed on raw replicate values without averaging. Data are shown as mean ± SEM. Shown p-values: L46S = 0.013, L46T = 0.003, L47Q = 0.086, S121N = 0.058. FIG. 14E: Amino acid variability across the HLA domains of 5,184 full-length HLA-A allele sequences from the IPD-IMGT / HLA database mapped to HLA-A*02:01:01:01 as reference. Yellow indicates conserved amino acid residue, whereas blue indicates an amino acid is different. The y-axis refers to 5184 unique HLA alleles and the x-axis refers to sequence of HLA. FIG. 14F: Number of amino acid changes per sequence position compared to HLA-A*02:01:01:01 for all 5,184 full-length HLA-A allele sequences aligned. Percentages of amino acid differences per domain relative to HLA-A*02:01:01: signal peptide (2.9%), al (7.2%), a2(9.2%), a3 (3.2%), transmembrane domain (2.9%), and cytoplasmic tail (3.7%). FIG. 14G: Live-cell imaging of HCC70 breast adenocarcinoma cells (transduced with mutant PIK3CA, HLA-A*03:01+, mCherry) co-cultured with TCR4-expressing T cells and CD8a mutants. The L46S (left panel) and S121N (right panel) mutations showed enhanced cytotoxicity compared to TCR4 and wild type CD8aP expressing T cells across multiple donors. (Full data set is shown in Extended data Fig.3h-l). Data are shown as mean ± SEM. Shown p-values: L46S = 0.023, S121N = 0.047. FIG.14H: Live-cell imaging of A375 cells (HLA-A*01:01+, expressing MAGE-A3) co-cultured with MAGE-A3 TCR expressing T cells and CD8a mutants. L46S (left panel)and S121N (right panel) CD8a variants enhanced cytotoxicity compared to wild-type CD8. Data are shown as mean ± SEM. Shown p-values: L46S = 0.009, S121N = 0.013.-8- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0038] FIGs. 15A-15G: CD8 mutants preserve TCR specificity while enhancing T cell activation. FIG. 15A: IFNy ELISpot assay for CD8 mutant 1G4 / TS T cells in response to NY-ESO-1 and various off-target peptides identified as reactive with the affinity-enhanced 1G4 / LY TCR. Despite enhanced on-target reactivity, CD8 mutants did not increase off-target reactivity compared to native 1G4 / TS T cells. NY-ESO-1 is the positive peptide control. PHA is assay positive control. MART-1 is negative peptide control. T2 cells and T cells alone are negative controls. FIG. 15B: IFNy ELISpot assays with MAGE-A3 TCR-expressing CD8 mutants co-cultured with T2 A*02:01 KO cells expressing HLA-A*01:01, pulsed with the MAGE-A3 on-target peptide and the Titin off-target peptide (to which a3a TCR is reactive). L46S and S121N mutations did not induce reactivity to Titin,demonstrating enhanced on-target cytotoxicity without cross-reactivity. In contrast, the a3a affinity enhanced TCR reacted strongly with Titin. MAGE-A3 is positive control peptide. PHA is assay positive control. T2 cells and T cells alone are negative controls. FIG. 15C: Alanine and Glycine scans of peptide. IFNy ELISpot assays with TCR4, TCR + CD8aP and TCR4 T cells expressing CD8 mutants co-cultured with COS7 cells (HLA-A*03+) pulsed with Alanine or Glycine substitution peptides. L46S and S121N mutants exhibited the same off-target reactivity asTCR4 or TCR4 + CD8aP expressing T cells, confirming that the CD8a mutations do not increase off-target responses, “x” position was not substituted as already the same amino acid. ALHGGWTTK is the positive control target peptide, and its sequence is also noted in the x axis. FIG. 15D: Structural representation of the L46S and S121N CD8a mutant in complex with HLA-A*02:01 (PDB: 1AKJ). Mutations were modeled in Coot.L46S introduces a hydrophobic-to-polar substitution at position 46 in the CDR1 loop, facilitating additional hydrogen bonds with K58 of P2M, likely contributing to the enhanced stability and affinity of the TCR / CD8 / pMHC complex. The S121N CD8a mutant (PDB:1AKJ) shows a longer side chain containing an amide group that forms a more stable hydrogen bond compared to the hydroxyl group of serine. FIG. 15E: Flow cytometry histogram plots of Jurkat NFAT-GFP reporter cell line expressing 1G4TS TCR and CD8 variants L46S or S121N. Baseline eGFP+ expressing is minimal, CD8a and 1G4 staining are shown. FIG. 15F:NF AT activation dose-response curve in reporter cells stimulated with titrated NY-ESO-1 peptide on T2 cells, for wild-type CD8 and mutants, illustrating leftward ECso shift for L46S and S121N mutants. FIG. 15G: Quantified ECso values (mean ± SD, n = 3) from FIG. 15E showing 1.5 and 2.6-fold enhanced peptide sensitivity for L46S and S121Nmutants, respectively, relative to wild type CD8.-9- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0039] FIGs. 16A-16E: Anti-Tumor efficacy of TCR-transduced CD8+ T Cells with CD8 variants targeting mutant PIK3CA in mice. FIG. 16A: Scheme of experiment evaluating the in vivo anti-tumor efficacy and safety of TCR-transduced CD8+ Tcells targeting mutant PIK3CA. Mice were sorted to the indicated treatment groupsafter subcutaneous implantation of HCC70-Mut PIK3CA tumors, which were allowed to grow to ~75 mm3in volume. Mice were treated with an intravenous injection of CD8+ Tcells transduced with either TCR4 + CD8aP, TCR4 + CD8 variant (L46S or S121N), negative control influenza (Flu)-specific TCR-transduced CD8+ T cells, or PBS control. FIG. 16B: TCR expression levels on CD8+ T cells expressing TCR4 + CD8aP or TCR4 + CD8 variants (L46S, S121N) were measured by flow cytometry, confirming comparable transduction efficiency across groups. FIG. 16C: Tumor volumes of mice bearing HCC70-Mut PIK3CA tumors after receiving either TCR4+CD8aP or TCR+ CD8 variant L46S or TCR4+ CD8 variant S121N or as a control an irrelevant A03 Flu TCR or PBS (n= 5 mice per group). Data are shown as mean ± SEM. Tumor volumes were measured twice a week. Bars indicate mean ± SEM. Statistical significance was determined using unpaired two-tailed t-tests. Tumors treated with the S121N mutant showed significantly reduced volumes compared with WT CD8 (p = 0.0376), whereas L46S-treated tumors showed a similar but not statistically significant trend (p = 0.08). FIG. 16D: Kaplan-Meyer survival curve of mice bearing HCC70-Mut PIK3CA tumors treated with either isolated CD8+transduced with TCR4 + CD8aP, TCR4 CD8 variant L46S, or TCR4 + CD8 variant S121N T cells (n = 5 per group). Mice were monitored daily until reaching endpoint tumor burden. Statistical analysis was performed using the Log-rank (Mantel-Cox) test. Both CD8 variants significantly prolonged survival compared with TCR4 + CD8ap. Median survival was 29 days for TCR + CD8ab and 36 days for the L46S and the S121N variant (L46S: p = 0.011; S121N: p = 0.0086). FIG. 16E: No significant weight loss was observed in any treatment group, indicating that the treatments were well-tolerated with no major toxicity. Data are shown as mean ± SEM.

[0040] FIGs. 17A-17H: Addition of CD8a and CD8«P does not improve the cytotoxicity of native TCRs. FIG. 17A: Table showing all TCRs used in this study and their respective affinity to p: MHC, their target and their HLA restriction. FIGs. 17B-17C: Live cell killing over 96 hours of A375 cells (NY-ESO+ / A02+ / mCherry+) co-culture with T cells expressing 1G4 / LY, 1G4 / TS, 1G4 / TS + CD8a, 1G4 / TS + CD8aP or untransduced T cells (UT) or no T cells for two donors. Data shown as mean ± SEM. FIGs. 17D-17F: Live cell killing of HCC70 cells (mutPIK3CA + / A03+ / mCherry+) co-culture with T cells expressing TCR4, TCR4 + CD8a, TCR4 + CD8aP or untransduced T cells (UT) or no T cells for three donors.-10- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443Data shown as mean ± SEM. FIGs. 17G-17H: Live cell killing of A375 cells (MAGE-A3 + / A01+ / mCherry+) co-culture with T cells expressing MAGE A3 TCR, MAGE. A3 TCR + CD8a, MAGE A3 TCR + CD8aP, a3a TCR or untransduced T cells (UT) or no T cells for two donors.[00411 FIGs. 18A-18D: Generation of Jurkat eGFP NF AT CD8a library and identification of 17 hits. FIG. 18A: Histograms showing GFP expression of Jurkat cells transduced with a NF AT eGFP reporter vector when rested or stimulated with anti CD3 antibody. Clones were generated by transduction and subsequent single cell sorting. Clone 10 (in green) was selected for further experiments. FIG. 18B: MOI calculation for CD8a library transduction. Transduction efficiency was evaluated using an antibody against the VP chain of 1G4 (clone H131). FIG. 18C: Flow cytometry histograms showing the expression of CD8a, 1G4 / TS TCR, and GFP in library transduced Jurkat eGFP transduced cells i) before library transduction in gray; ii) after initial transduction in green; and iii) after magnetic activated bead separation in blue. FIG. 18D: Heat Map showing the relative fraction of enriched library clones for three independent screens per sorting round. Dark blue indicates highly enriched variants compared to baseline. The 17 clones depicted on the Y axis were statistically significantly enriched across the three rounds of screens.

[0042] FIGs. 19A-19F: Live cell imaging screens of native TCRs and CD8 screening hits validate some CD8 hits in two HLA / TCR pairs across multiple donors. FIG. 19A: Live cell killing assay of A375 cells co-cultured with either untransduced T cells, T cells expressing 1G4 / LY, 1G4 / TS, 1G4 / TS+ CD8a, 1G4 / TS + CD8aP, or 1G4 / TS and identified CD8 variants from Figure Ih. 3 different donors are shown. Raw confluence values were exported and analyzed in R (v4.3.3). Each condition included three technical replicates per donor. Data were analyzed by unpaired two-sample t-tests (Welch’s correction) comparing each condition to the control (1G4 / TS + CD8aP or 1G4 / TS). Tests were performed on raw replicate values without averaging. Data are shown as mean ± SEM. Data visualization was done with ggplot2 (v3.5.1).FIG. 19B: Live cell killing assay of A375 co-cultured with either untransduced T cells, T cells expressing 1G4 / LY, 1G4 / TS, 1G4 / TS+ CD8a, 1G4 / TS + CD8aP, or 1G4 / TS and CD8 variants. Top variants were picked from data in FIGs. 19A-19C above. 3 different donors are shown. Data are shown as mean ± SEM. FIG. 19C: Live cell killing assay of HCC70 cells co-cultured with either untransduced T cells, T cells expressing TCR4, TCR4 + CD8a, TCR4 + CD8aP and TCR4 + identified CD8 variants from Figure Ih. 3 different donors are shown. Data are shown as mean ± SEM. FIG. 19D: Live cell killing assay of HCC70 cells co-cultured with either untransduced T cells, T cells expressing TCR4, TCR4 + CD8a, TCR4 + -11- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443CD8aP and TCR4 + identified CD8 variants. Top variants were identified to enhance cytotoxicity in FIG. 19C above. 2 different donors are shown. Data are shown as mean ± SEM. FIG. 19E: Heat map shows normalized confluence values (%) at 96 h for all donors and CD8 mutants in the initial hit validation screens using 1G4 / TS and TCR4 model. Each cell represents the mean of three technical replicates per donor, normalized to the maximum confluence within that donor (100 % = no killing). Lower percentages indicate higher cytotoxicity. FIG. 19F: Heat map shows normalized confluence values (%) at 96 h for additional donors and selected CD8 mutants. Each cell represents the mean of three technical replicates per donor, normalized to the maximum confluence within that donor (100 % = no killing). Lower percentages indicate higher cytotoxicity. Statistical significance was determined per donor using unpaired Welch’s t-tests comparing each mutant to the + CD8aP control. Variants with p < 0.05 are indicated on the heat map (asterisks). Data visualization was done with ggplot2 (v3.5.1). X-ed cell means this condition was not tested during this experiment.10043] FIG. 20A: A total of 6,375 full-length HLA-B sequence were retrieved from IMGT / HLA database and aligned to HLA-B*07:02:01:01 using Clustal Omega. Yellow represents a conserved amino acid, blue represents an amino acid difference. FIG. 20B: The number of amino acid changes to the control sequence HLA-B*07:02:01:01 were plotted per sequence position. The a3 domain of HLA-B show lowest variation compared to the al and a2 domains. FIG. 20C: A total of 4,268 full-length HLA-C sequence were retrieved from IMGT / HLA database and aligned to HLA-C*01:02:01:01 using Clustal Omega. Yellow represents a conserved amino acid, blue represents an amino acid difference. FIG. 20D: The number of amino acid changes to the control sequence HLA-C*01:02:01:01 were plotted per sequence position. The a3 domain of HLA-C show lowest variation compared to the al and a2 domains.

[0044] FIGs. 21A-21D: Enhanced tetramer binding of CD8 variants across donors and TCR / HLA pairs. FIG. 21A: NY-ESO-1 / A02 tetramers on T cells expressing 1G4 / LY or 1G4 / TS + CD8aP or mutant L46S or S121N CD8 variants, showing consistently increased tetramer binding frequencies for T cells expressing either CD8 variant, compared to CD8 wild type in donor 1. FIG. 21B: MAGE-A3 / A01 tetramers on T cells expressing a3a TCR or MAGE A3 TCR + CD8aP or mutant L46S or S121N CD8 variants, showing consistently increased tetramer binding frequencies for T cells expressing either CD8 variant, compared to CD8 wild type in donor 1. FIG. 21C: NY-ESO-1 / A02 tetramers on T cells expressing 1G4 / LY or 1G4 / TS + CD8aP or mutant L46S or S121N CD8 variants, showing consistently -12- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443increased tetramer binding frequencies for T cells expressing either CD8 variant, compared to CD8 wild type in donor 2. FIG. 21D: MAGE-A3 / A01 tetramers on T cells expressing a3a TCR or MAGE A3 TCR + CD8aP or mutant L46S or S121N CD8 variants, showing consistently increased tetramer binding frequencies for T cells expressing either CD8 variant, compared to CD8 wild type in donor 2.

[0045] FIGs. 22A-22D: CD8aa reduces off target reactivity of an affinity-enhanced TCR while maintaining some on target efficacy. FIG. 22A: Schematic overview of the generation of CD8- / - (CD8a7") and CD8aa (CD8P7") T cells using CRISPR / Cas9, followed by MACS separation. This strategy allows for the generation of pure CD8aa and CD8- / -populations. FIG. 22B: Representative flow plot showing the expression of CD8a and CD8P in CD8- / - and CD8aa 1G4 / LY T cells, with TCR expression remaining comparable across all conditions. FIG. 22C: CD8aa exhibit reduced off-target reactivity compared to affinity-enhanced CD8 lG4LY-expressing T cells (lower panel). NY-ESO-1 is the positive peptide control. PHA is assay positive control. MART-1 is negative peptide control. T2 cells and T cells alone are negative controls. Data are shown as mean ± SD. FIG. 22D: Live-cell cytotoxicity analysis of A375 (GFP-NLS) and SKMEL-37 (GFP-NLS) melanoma cells cocultured with isolated CD8+untransduced (UT) CD8 1G4L / Y, 1G4 / LY CD8- / -, and 1G4 / LY CD8aa T cells for 4 days. Data are shown as mean ± SEM.

[0046] FIGs. 23A-23C: Overexpression of CD8a leads to indiscriminate tetramer staining. FIG. 23A: Live-cell killing analysis of A375 (GFP-NLS) and SKMEL-37 (GFP-NLS) melanoma cells co-cultured with isolated CD8+ untransduced, CD8 1G4 / LY, CD8 1G4 / LY + CD8a, CD8aa 1G4LY, or CD8aa 1G4LY + CD8a T cells for 4 days. CD8a overexpression did not significantly enhance cytotoxicity in 1G4LY T cells. Data are shown as mean ± SEM. FIG. 23B: Flow cytometry plots showing irrelevant WT1 / A02 tetramer staining for isolated CD8+untransduced T cells, CD8 1G4 / LY, CD8 1G4 / LY + CD8a, CD8aa 1G4 / LY or CD8aa 1G4 + CD8a. FIG. 23C: Frequency of tetramer positive cells of cells as described in above FIG. 23B for multiple tetramers. NY-ESO-1 is on target tetramer; all other tetramers are irrelevant tetramers. Upon SKI antibody (anti CD8a antibody) blockade tetramer staining is reduced.DETAILED DESCRIPTION

[0047] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.-13- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[0048J In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel etal. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson etal. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U. S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg etal. eds (1996) Weir’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).

[0049] Typical therapeutic anti-cancer monoclonal antibodies (mAb), like those that bind to CD 19, and Chimeric Antigen Receptor T cells (CAR T cells) recognize cell surface proteins. Cell surface proteins constitute only a tiny fraction of the cellular protein content. Most mutated or oncogenic tumor associated proteins are typically nuclear or cytoplasmic, preventing use of canonical anti-cancer mAbs and CAR-T cells to target such mutated or oncogenic tumor associated proteins. In certain instances, these intracellular proteins can be degraded in the proteasome, processed, and presented on the cell surface by MHC class I molecules as T cell epitopes that are recognized by T-cell receptors (TCRs). These peptides can be derived from proteins from any cellular location, thus vastly expanding the repertoire of potentially targetable cancer antigens. However, TCR cross-reactivities with presented peptides that have similar amino acid sequences are frequently observed. One challenge of TCR-based therapies is that it is extremely difficult to predict off-target reactivities that can lead to toxicities or to modulate such detrimental cross-reactions. Therefore, methods to-14- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443modulate TCR specificity are increasingly useful in, for example, the treatment of subjects in need thereof (e.g., subjects with cancer) using TCR-based therapies.

[0050] The CD8 co-receptor stabilizes the TCR / peptide-MHC complex by interacting with MHC class I molecules on target cells (FIG. 1). The immune system favors lower-affinity TCRs (Kd > 6pM) to prevent autoimmunity while maintaining broad antigen reactivity. CD8 typically enhances TCR signaling, especially for lower-affinity TCRs. Enhancing TCR affinity by altering the TCR sequence can improve T cell responses to weak antigens. The affinity enhanced 1G4 TCR (A95: LY), which targets theNY-ESO-1 cancer-testis antigen, is currently in clinical trials. This modified 1G4 TCR operates independently of CD8, allowing both CD8 and CD4 T cells to be activated. However, this CD8-independent behavior increases off-target reactivity, as thymic negative selection may no longer safeguard against autoimmunity.

[0051] The present disclosure demonstrates, among other things, that through mutation of CD8a, off-target reactivity with certain CD8-dependent peptides in CD4 and CD8 cells could be reduced without sacrificing on-target cytotoxicity. Accordingly, technologies of the present disclosure provide, among other things, engineered immune cells with improved TCR specificity and decreased associated toxicity of TCR-T cells. The compositions and methods disclosed herein do not rely on altering the TCR sequence itself, and maintain the cytotoxic anti-cancer potential of these cells. Such an approach might be applied generally to other therapeutic TCRs without the need to change the underlying TCR therapeutic agent.Definitions

[0052] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like.Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0053] As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%,-15- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443up 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, within 5-fold, or within 2-fold, of a value.

[0054] As used herein, the “administration” of an agent (e.g., engineered immune cells as described herein or drug) to a subject includes any route of introducing or delivering to a subject the agent to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another.

[0055] As used herein “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises a cell type including, for example, TCR (i.e., heterologous T-cell receptor) modified lymphocytes (e.g., eTCR T cells and caTCR T cells). In another embodiment, the adoptive cell therapeutic composition comprises T cells. In another embodiment, T-cells form the adoptive cell therapeutic composition.

[0056] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide are in the D form, and a second plurality of amino acids are in the L form.

[0057] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.-16- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[0058| As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. As used herein, “antibodies” (includes intact immunoglobulins) and “antigen binding fragments” specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M-l greater, at least 104M-l greater or at least 105M-l greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

[0059] More particularly, an antibody refers to a polypeptide ligand comprising at least a light chain immunoglobulin variable region or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. Typically, an immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (1) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementaritydetermining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat eta!.. Sequences of Proteins of Immunological Interest, U. S. Department of Health and Human Services, 1991, which is hereby incorporated by reference). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, -17- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443largely adopt a P-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the P-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.[0060| The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs). An antibody or antigen binding fragment thereof specifically binds to an antigen.

[0061] As used herein, an “antigen” refers to a molecule to which an immunoglobulin-related composition (e.g., antibody or antigen binding fragment thereof or T Cell Receptor) can selectively bind. The target antigen may be a protein, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen may be a peptide / MHC complex. An antigen may also be administered to an animal to generate an immune response in the animal.

[0062] The term “antigen binding fragment” refers to a fragment of the whole immunoglobulin structure which possesses a part of a polypeptide responsible for binding to antigen. Examples of the antigen binding fragment include scFv, (scFv)2, scFvFc, Fab, Fab' and F(ab')2, but are not limited thereto.

[0063] By “binding affinity” is meant the strength of the total noncovalent interactions between a single binding site of a molecule (e.g., an immunoglobulin-related composition, TCR) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Ka). Affinity can be measured by standard methods known in the art, including those described herein. A low-affinity complex contains an immunoglobulin-related composition that generally tends to dissociate readily from the antigen, whereas a high-affinity complex contains an immunoglobulin-related composition that generally tends to remain bound to the antigen for a longer duration.-18- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[0O64| As used herein, the term “biological sample” means sample material derived from living cells. Biological samples may include tissues, cells, protein or membrane extracts of cells, and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids present within a subject. Biological samples of the present technology include, but are not limited to, samples taken from breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, thymus, blood, hair, buccal, skin, serum, plasma, CSF, semen, prostate fluid, seminal fluid, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from non-diseased individuals, as controls or for basic research. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a tissue sample obtained by needle biopsy.

[0065] As used herein, a “cancer” is a disease state characterized by the presence in a subject of cells demonstrating abnormal uncontrolled replication and in some aspects, the term may be used interchangeably with the term “tumor.” The term “cancer or tumor antigen” refers to an antigen known to be associated and expressed in a cancer cell or tumor cell or tissue, and the term “cancer or tumor targeting antibody” refers to an antibody that targets such an antigen. In some embodiments, the cancer or tumor antigen is not expressed in a non-cancer cell or tissue. In some embodiments, the cancer or tumor antigen is expressed in a non-cancer cell or tissue at a level significantly lower compared to a cancer cell or tissue.

[0066] In some embodiments, the cancer is selected from: circulatory system, for example, heart (sarcoma [angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma], myxoma, rhabdomyoma, fibroma, and lipoma), mediastinum and pleura, and other intrathoracic organs, vascular tumors and tumor-associated vascular tissue; respiratory tract, for example, nasal cavity and middle ear, accessory sinuses, larynx, trachea, bronchus and lung such as small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; gastrointestinal system, for example, esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,-19- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443leiomyosarcoma), gastric, pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); gastrointestinal stromal tumors and neuroendocrine tumors arising at any site; genitourinary tract, for example, kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and / or urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); liver, for example, hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma, pancreatic endocrine tumors (such as pheochromocytoma, insulinoma, vasoactive intestinal peptide tumor, islet cell tumor and glucagonoma); bone, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system, for example, neoplasms of the central nervous system (CNS), primary CNS lymphoma, skull cancer (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain cancer (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); reproductive system, for example, gynecological, uterus (endometrial carcinoma), cervix (cervical carcinoma, pre- tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), placenta, vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma) and other sites associated with female genital organs;, penis, prostate, testis, and other sites associated with male genital organs; hematologic system, for example, blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma]; oral cavity, for example,-20- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443lip, tongue, gum, floor of mouth, palate, and other parts of mouth, parotid gland, and other parts of the salivary glands, tonsil, oropharynx, nasopharynx, pyriform sinus, hypopharynx, and other sites in the lip, oral cavity and pharynx; skin, for example, malignant melanoma, cutaneous melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids; adrenal glands: neuroblastoma; and other tissues comprising connective and soft tissue, retroperitoneum and peritoneum, eye, intraocular melanoma, and adnexa, breast, head or neck, anal region, thyroid, parathyroid, adrenal gland and other endocrine glands and related structures, secondary and unspecified malignant neoplasm of lymph nodes, secondary malignant neoplasm of respiratory and digestive systems and secondary malignant neoplasm of other sites. In some embodiments, the cancer is a colon cancer, colorectal cancer or rectal cancer. In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a pancreatic cancer. In some embodiments, the cancer is an adenocarcinoma, an adenocarcinoma, an adenoma, a leukemia, a lymphoma, a carcinoma, a melanoma, an angiosarcoma, or a seminoma.

[0067] In some embodiments, the cancer is a solid tumor. In other embodiments, the cancer is not a solid tumor. In some embodiments, the cancer is from a carcinoma, a sarcoma, a myeloma, a leukemia, or a lymphoma. In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some embodiments, the cancer is a relapsed cancer. In some embodiments, the cancer reaches a remission, but can relapse. In some embodiments, the cancer is unresectable.

[0068] As used herein, the term “conservative sequence modification” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of a particular polypeptide comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the presently disclosed technologies 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 and glutamic acid; and 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 -21- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443polarity: 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 certain region can be replaced with other amino acid residues from the same group and the altered protein can be tested for retained function (i.e., the functions set forth in (c) through (1) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a specified sequence are altered.

[0069] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0070] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease and / or condition described herein or one or more signs or symptoms associated with a disease and / or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0071] As used herein, the term “epitope” means an antigenic determinant capable of specific binding to an immunoglobulin-related composition such as an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific -22- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. In some embodiments, an “epitope” is a region of the target antigen to which TCR compositions of the present technology specifically bind. In some embodiments, the epitope is a conformational epitope or a non-conformational epitope. Epitope mapping can be performed by methods known in the art.

[0072] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0073] As used herein, an “expression control sequence” refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to encompass, at a minimum, any component whose presence is essential for expression, and can also encompass an additional component whose presence is advantageous, for example, leader sequences.

[0074] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0075] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab1) (bivalent) regions, wherein each (ab1) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the-23- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0076] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0077] The term “HLA-A2”, as used herein, representatively refers to the subtypes, examples of which include, but are not limited to, HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*02:05, HLA-A*02:06, HLA-A*02:07, HLA-A*02:10, HLA-A*02:ll, HLA-A*02:13, HLA-A*02:16, HLA-A*02:18, HLA-A*02:19, HLA-A*02:28 and HLA-A*02:50.

[0078] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by =HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0079] As used herein, the terms “identical” or percent “identity”, when used in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or -24- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443nucleotides that are the same (i.e., about 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding a TCR or CD8 molecule as described herein or amino acid sequence of a TCR or CD8 molecule as described herein)), when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (e.g., NCBI web site). Such sequences are then said to be “substantially identical.” This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and / or additions, as well as those that have substitutions. In some embodiments, identity exists over a region that is at least about 8, 9, 10, 11, 12, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more amino acids in length. In some embodiments, identity exists over a region that is at least about 21, 24, 27, 30, 33, 36, 45, 60, 75, 150, 225, 300, 450, 600, 750, 900, 1050, 1200, 1350, 1500, 1650, 1800, 1950, 2100, 2250, 2400, 2550, 2700, 2850, 3000 or more nucleotides in length.

[0080] As used herein, the term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. As used herein, the term “engineered immune cell” refers to an immune cell that is genetically modified, and in particular, wherein the immune cell is a T cell. As used herein, the term “native immune cell” refers to an immune cell that naturally occurs in the immune system.

[0081] As used herein, the term “increase” means to alter positively by at least about 5%, including, but not limited to, alter positively by about 5%, by about 10%, by about 25%, by about 30%, by about 50%, by about 75%, or by about 100%.

[0082] As used herein, the terms “individual”, “patient”, or “subject” can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient or subject is a human.

[0083] As used herein, the term “MHC” refers to the Major Histocompability Complex, which is defined as a set of gene loci specifying major histocompatibility antigens. The term “HLA” as used herein will be understood to refer to Human Leukocyte Antigens, which is defined as the histocompatibility antigens found in humans. As used herein, “HLA” is the human form of “MHC”.-25- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0084] As used herein, the terms “MHC light chain” and “MHC heavy chain” refer to portions of the MHC molecule. Structurally, class I molecules are heterodimers comprised of two noncovalently bound polypeptide chains, a larger “heavy” chain (a) and a smaller “light” chain (P2-microglobulin or 32m). The polymorphic, polygenic heavy chain (45 kDa), encoded within the MHC on chromosome six, is subdivided into three extracellular domains (designated 1, 2, and 3), one intracellular domain, and one transmembrane domain. The two outermost extracellular domains, 1 and 2, together form the groove that binds antigenic peptide. Thus, interaction with the TCR occurs at this region of the protein. The 3 domain of the molecule contains the recognition site for the CD8 protein on the CTL; this interaction serves to stabilize the contact between the T cell and the APC. The invariant light chain (12 kDa), encoded outside the MHC on chromosome 15, consists of a single, extracellular polypeptide. The terms “MHC light chain”, “P-2-microglobulin”, and “ 2m” may be used interchangeably herein.

[0085] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. For example, a monoclonal antibody can be an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including, e.g., but not limited to, hybridoma, recombinant, and phage display technologies. For example, the monoclonal antibodies to be used in accordance with the present methods may be made by the hybridoma method first described by Kohler et aL, Nature 256:495 (1975), or may be made by recombinant DNA methods (See, e.g, U. S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et aL, J. Mol. Biol. 222:581-597 (1991), for example.-26- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0086] As used herein, "operably linked" with reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a nucleic acid encoding a leader peptide can be operably linked to a nucleic acid encoding a polypeptide, whereby the nucleic acids can be transcribed and translated to express a functional fusion protein, wherein the leader peptide affects secretion of the fusion polypeptide. In some instances, the nucleic acid encoding a first polypeptide (e.g., a leader peptide) is operably linked to nucleic acid encoding a second polypeptide and the nucleic acids are transcribed as a single mRNA transcript, but translation of the mRNA transcript can result in one of two polypeptides being expressed. For example, an amber stop codon can be located between the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide, such that, when introduced into a partial amber suppressor cell, the resulting single mRNA transcript can be translated to produce either a fusion protein containing the first and second polypeptides, or can be translated to produce only the first polypeptide. In another example, a promoter can be operably linked to nucleic acid encoding a polypeptide, whereby the promoter regulates or mediates the transcription of the nucleic acid.

[0087] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0088] As used herein, the term “polyclonal antibody” means a preparation of antibodies derived from at least two (2) different antibody-producing cell lines. The use of this term includes preparations of at least two (2) antibodies that contain antibodies that specifically bind to different epitopes or regions of an antigen.

[0089] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be singlestranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one-27- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0090] As used herein, the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.

[0091] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (nonrecombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0092] As used herein, “regulatory region” of a nucleic acid molecule means a cis- acting nucleotide sequence that influences expression, positively or negatively, of an operably linked gene. Regulatory regions include sequences of nucleotides that confer inducible (i.e., require a substance or stimulus for increased transcription) expression of a gene. When an inducer is present or at increased concentration, gene expression can be increased. Regulatory regions also include sequences that confer repression of gene expression i.e., a substance or stimulus decreases transcription). When a repressor is present or at increased concentration, gene expression can be decreased. Regulatory regions are known to influence, modulate or control many in vivo biological activities including cell proliferation, cell growth and death, cell differentiation and immune modulation. Regulatory regions typically bind to one or more trans-acting proteins, which results in either increased or decreased transcription of the gene.

[0093] Particular examples of gene regulatory regions are promoters and enhancers.Promoters are sequences located around the transcription or translation start site, typically positioned 5' of the translation start site. Promoters usually are located within 1 Kb of the translation start site, but can be located further away, for example, 2 Kb, 3 Kb, 4 Kb, 5 Kb or more, up to and including 10 Kb. Enhancers are known to influence gene expression when -28- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443positioned 5' or 3' of the gene, or when positioned in or a part of an exon or an intron.Enhancers also can function at a significant distance from the gene, for example, at a distance from about 3 Kb, 5 Kb, 7 Kb, 10 Kb, 15 Kb or more. Regulatory regions also include, but are not limited to, in addition to promoter regions, sequences that facilitate translation, splicing signals for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA and, stop codons, leader sequences and fusion partner sequences, internal ribosome binding site (IRES) elements for the creation of multigene, or polycistronic, messages, polyadenylation signals to provide proper polyadenylation of the transcript of a gene of interest and stop codons, and can be optionally included in an expression vector.

[0094] As used herein, the term “sample” refers to clinical samples obtained from a subject. In certain embodiments, a sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, bodily fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0095] As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.

[0096] As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.

[0097] As used herein, “specifically binds” refers to a molecule (e.g., an immunoglobulin-related composition) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule, as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about 104M, IO5M, 106M, 107M, 10"8M, 109M, 1010M, 10 " M, or 10l2M. The term “specifically binds” may also refer to binding where a molecule (e.g., TCR) binds to a particular target molecule or complex (e.g., peptides presented on cell surfaces in the context-29- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443of major histocompability complexes), without substantially binding to any other molecule or complex.

[0098] As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.

[0099] As used herein, "synthetic," with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant methods and / or by chemical synthesis methods. As used herein, "production by recombinant means by using recombinant DNA methods" means the use of the well-known methods of molecular biology for expressing proteins encoded by cloned DNA.

[0100] As used herein, the term “T-cell” includes naive T cells, CD4+ T cells, CD8+ 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), activated T cells, anergic T cells, tolerant T cells, chimeric B cells, Regulatory T cells (also known as suppressor T cells), Natural killer T cells, Mucosal associated invariant T cells, and y6 T cells, and antigen-specific T cells.

[0101] As used herein, “T cell receptor” or “TCR”, is a protein complex found on the surface of T cells that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex molecules. TCR is composed of two disulfide-linked protein chains. Cells expressing a TCR containing the highly variable alpha (a) and beta (P) chains are referred to as aP T cells. Cells expressing an alternate TCR, formed by variable gamma (y) and delta (8) chains, are referred to as y6 T cells. When the TCR engages with antigenic peptide and MHC (peptide / MHC or pMHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, coreceptors, specialized adaptor molecules, and activated or released transcription factors. In some embodiments, a TCR is a native T cell receptor that is endogenous to the immune cells. In some embodiments, a TCR is an artificial receptor that mimics native TCR function, i.e., recognizing peptide antigens of key intracellular proteins in the context of MHC on the cell surface.

[0102] The term “TCR-associated signaling molecule” refers to a molecule having a cytoplasmic immunoreceptor tyrosine-based activation motif (IT AM) that is part of the TCR-CD3 complex. TCR-associated signaling molecules include CD3ys, CD36s, and CD3ζ (also known as (, CD3 or TCRQ.-30- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0103] As used herein, the terms “mimic TCR,” “TCRm” or “TCR-like” refers to an artificial receptor that mimics native TCR function (i.e., recognizing peptide antigens of key intracellular proteins in the context of MHC on the cell surface). Examples of different types mimic TCRs (e.g., TCRm Abs, TCRm BITEs, TCRm CARs, and ImmTACs) are described in detail in Jones et al., Front. Immunol., 25 January 2021, doi.org / 10.3389 / fimmu.2020.585385, the contents of which are incorporated by reference herein in its entirety.10104] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0105] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0106] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0107] As used herein, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or fragment thereof can be introduced, typically by restriction digest and ligation. Reference to a vector also includes those vectors that contain nucleic acid encoding a polypeptide. The vector is used to introduce the nucleic acid encoding the polypeptide into the host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. The vectors typically remain episomal, but can be designed to effect integration of a gene or portion thereof into a chromosome of the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. Selection and use of such vehicles are well known to those of skill in the art. A vector also includes "virus vectors" or "viral vectors." Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or -31- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443shuttles) the exogenous genes into cells. As used herein, an "expression vector" includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.T-Cell Receptors (TCRs}

[0108] T cells are part of the adaptive immune system and target cancerous and / or infected cells through T-cell receptors (TCRs). T cell receptors are protein complexes found on the surface of T cells and are responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex molecules. TCRs are composed of two disulfide-linked protein chains. Cells expressing a TCR containing the highly variable alpha (a) and beta (P) chains are referred to as aP T cells. Cells expressing an alternate TCR, formed by variable gamma (y) and delta (8) chains, are referred to as y6 T cells. Without wishing to be bound by any one theory, variable chains allows for the recognition of many different peptides (e.g., target antigens, including, for example, tumor antigens) presented on MHC molecules.

[0109] There are two major groups of MHC molecules, class I (MHC I) and class II (MHC II). MHC I molecules interact with CD8+ T cells and MHC II molecules interact with CD4+ T cells. MHC I molecules present peptides, typically 8-12 amino acids long, derived from proteins in any cellular compartment. Presented peptides (e.g., target antigens, including, for example, tumor antigens) are generated as a result of protein degradation through the proteasome, cleavage by aminopeptidases, and transport to the Endoplasmic Reticulum (ER) via the transporter associated with antigen processing (TAP). Peptides are then loaded onto MHC I molecules, resulting in a peptide-MHC complex. These peptide-MHC (pMHC) complexes are then shuttled to the cell surface where they are presented to CD8+ T cells and are recognized by TCRs. When TCRs engage with antigenic peptide and MHC (peptide / MHC), the T cell is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor-32- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443molecules, and activated or released transcription factors, ultimately resulting in immune-mediated cell death.10110] Accordingly, in some embodiments, a TCR as disclosed herein binds to a target antigen. In further embodiments, a target antigen is a tumor antigen presented in the context of a MHC I molecule. In some such embodiments, a tumor antigen is or comprises, for example, Tyrosinase, NY-ESO-1, CD277-mediated presentation, MAGE-A4, WT1, MAGE-A10, PRAME, EBV LMP2, MAGE-A1, HA-1, HERV-E, CMV pp65, HBV, TRAIL-DR4, HIV SL9, or AFP. In some embodiments, a MHC I molecule is a HLA-A, HLA-B, or HLA-C molecule. In some embodiments, a target antigen comprises a tumor antigen presented in the context of a HLA-A2 molecule.

[0111] In some embodiments, a TCR binds to a target antigen that is expressed by a tumor cell (e.g., a tumor antigen). In some embodiments, a TCR binds to a target antigen that is expressed on the surface of a tumor cell as part of the pMHC complex. In some such embodiments, a target antigen expressed on the surface of a tumor cell as part of the pMHC complex is expressed intracellularly when not as part of the pMHC complex. In some embodiments, the MHC protein is a MHC class I protein. In some embodiments, the MHC class I protein is a HLA-A, HLA-B, or HLA-C molecule. In some embodiments, a HLA-A molecule is a HLA-A2 molecule.

[0112] In some embodiments, a TCR is a native T cell receptor that is endogenous to an immune cell (e.g., T cell). In some such embodiments, a native TCR is or comprises, for example, lG4-a95TS (also referred to as lG4a95TS, native 1G4 TCR, and native 1G4; see, e.g., Robbins PF etal., Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol. 2008;180(9):6116-6131.doi: 10,4049 / j immunol.180.9.6116).

[0113] In some embodiments, a TCR is a non-native TCR. In some such embodiments, a nonnative TCR is an engineered TCR that binds to a target antigen (e.g., tumor antigen). In some embodiments, an engineered TCR is an affinity enhanced TCR. In some such embodiments, an affinity enhanced TCR is or comprises, for example, 1G4 TCR (also referred to as 1G4-a95LY and lG4a95LY; see, e.g., Robbins PF etal., Single and dual amino acid substitutions in TCR CDRs can enhance antigen-specific T cell functions. J Immunol. 2008;180(9):6116-6131. doi: 10,4049 / j immunol.180.9.6116 ).

[0114] In some embodiments, a TCR is a mimic TCR. A mimic TCR is, for example, an artificial receptor that mimics native TCR function (i.e., recognizing peptide antigens of key intracellular proteins in the context of MHC on the cell surface).-33- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[0115| In some embodiments, a TCR is 1G4, DMF5, TCR4, MAGE A3, or A6 TCR. In some embodiments, a TCR is an affinity enhanced IG4 (e.g., 1G4 / LY, 1G4 / TS), DMF5, TCR4, MAGE A3, or A6 TCR.

[0116] In some embodiments, a TCR comprises a high binding specificity and / or high binding affinity to a target antigen. For example, in some embodiments, a TCR binds to a particular target antigen with a dissociation constant (Ka) of about 1 x 10’5M or less. In certain embodiments, the Ka is about 5 x 10'6M or less, about 1 x 10'6M or less, about 5 x 10'7M or less, about 1 x 107M or less, about 5 x 10'8M or less, about 1 x 10'8M or less, about 5 xlO'9or less, about 4 x 10'9or less, about 3 x 10'9or less, about 2 x 10'9or less, or about 1 x 10'9M or less. In certain non-limiting embodiments, the Ka is from about 3 x 10'9M or less. In certain non-limiting embodiments, the Ka is from about 3 x 10'9to about 2 x IO'7

[0117] Binding of a TCR of the present disclosure can be assessed by, for example, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISpot) assay, 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, a TCR 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).

[0118] The presently disclosed subject matter also provides nucleic acids encoding TCRs as described herein or a functional portion thereof. In some embodiments, nucleic acids encoding TCRs are isolated nucleic acid molecules. In certain embodiments, a nucleic acid molecule comprises a nucleic acid sequence that encodes a functional portion of a TCR construct. As used herein, the term “functional portion” refers to any portion, part or fragment of a TCR, which portion, part, or fragment retains the biological activity of the parent TCR. For example, functional portions encompass the portions, parts, or fragments of a TCR that retains the ability to recognize peptides presented on the cell surface in the context of a MHC (e.g., MHC I) to a similar, same, or even higher extent as the parent TCR. In certain embodiments, a -34- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443nucleic acid molecule encoding a functional portion of a target-antigen specific TCR can encode a protein comprising, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, or more of the parent TCR.

[0119] Additionally or alternatively, in certain embodiments, a nucleic acid comprises a nucleic acid sequence encoding a TCR and a first reporter or selection marker (e.g., GFP, puromycin resistance). In some such embodiments, the nucleic acid encoding the TCR and first reporter or selection marker are separated by a nucleic acid sequence encoding an Internal Ribosomal Entry Site (IRES). In some such embodiments, the TCR and first reporter or selection marker are linked by a self-cleaving linker, such as a P2A linker. In certain embodiments, a heterologous TCR and a reporter or selection marker (c.g, GFP, puromycin resistance) are expressed as two separate polypeptides.

[0120] Additionally or alternatively, in certain embodiments, a nucleic acid comprises a nucleic acid sequence encoding a TCR and a CD8a polypeptide (as described elsewhere herein). In some such embodiments, the TCR and a CD8a polypeptide are linked by a selfcleaving linker, such as a P2A linker. In certain embodiments, a heterologous TCR and a CD8a polypeptide are expressed as two separate polypeptides. In some such embodiments, the nucleic acid sequence encoding a TCR and a CD8a polypeptide are separated by a nucleic acid sequence encoding an Internal Ribosomal Entry Site (IRES).

[0121] Additionally or alternatively, in some embodiments, a heterologous nucleic acid comprising a nucleic acid sequence encoding a CD8a polypeptide (as described elsewhere herein) and / or a TCR of the present disclosure is operably linked to an inducible promoter. In some embodiments, a heterologous nucleic acid comprising a nucleic acid sequence encoding a CD8a gene (as described elsewhere herein) and / or a TCR of the present disclosure is operably linked to a constitutive promoter.

[0122] Among other things, technologies of the present disclosure provide engineered immune cells (e.g., engineered cytotoxic T cells, engineered CD4+ helper T cells) that comprise a TCR that binds to a target antigen and / or a nucleic acid encoding the TCR. In some embodiments, engineered immune cells of the present disclosure express CD8a (as described elsewhere herein) and / or a TCR (e.g., a native TCR, a non-native TCR, mimic TCR). In certain embodiments, engineered immune cells can be transduced with a TCR construct such that the cells express the TCR. In some embodiments, a coding sequence of a TCR is endogenously present in an engineered immune cell. In some embodiments, a coding sequence of a TCR is exogenously provided to an engineered immune cell via a nucleic acid vector, such as a -35- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443retroviral vector. Technologies of the present disclosure also provide, among other things, methods of using such cells for the treatment of cancer.CD8a

[0123] As used herein, the terms “CD8 alpha”, “CD8a”, “CD8a polypeptide” “Leu2”, and “P32” refer to a cell surface glycoprotein found on most cytotoxic T cells that mediates efficient cell-cell interactions within the immune system. The encoded protein also acts as a co-receptor with TCRs on the T cell to recognize antigens displayed by an antigen presenting cell in the context of class I MHC molecules. The CD8 co-receptor functions as either a homodimer, comprised of two alpha (a) chains, or as a heterodimer, comprised of one alpha (a) and one beta (P) chain. Non-limiting examples of this polypeptide or underlying gene may be found under the Gene Cards IDs: GC02M086907 GC02M087305, GC02M086986, GC02M086923, GC02M086865 (retrieved from https: / / www.genecards.org / cgi-bin / carddisp.pl?gene=CD8A#summaries), HGNC: 1706 (https: / / www.genenames. Org / data / gene-symbol-report / #l / hgnc_id / 1706), NCBI Entrez Gene: 925 (https: / / www.ncbi.nlm.nih.gov / gene / 925), Ensembl: ENSG00000153563 (https: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000153563;r=2:867846 10-86808396), OMIM®: 186910 (https: / / omim.org / entry / 186910), or UniProtKB / Swiss-Prot: P01732 (https: / / www.uniprot.org / uniprot / P01732), which are incorporated by reference herein.

[0124] The presently disclosed subject matter also provides nucleic acids encoding CD8a polypeptides as described herein or a functional portion thereof. As used herein, the term “functional portion” refers to any portion, part or fragment of a CD8a polypeptide, which portion, part, or fragment retains the biological activity of the parent CD8a polypeptide. For example, functional portions encompass the portions, parts, or fragments of a CD8a polypeptide that retains the ability to bind to MHC relative to the wild-type CD8a polypeptide to a similar, same, or even higher extent as the parent CD8a polypeptide. In certain embodiments, a nucleic acid molecule encoding a functional portion of a CD8a polypeptide can encode a polypeptide comprising, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, and about 95%, or more of the parent CD8a polypeptide. In some embodiments, nucleic acids encoding CD8a polypeptides are isolated nucleic acids. In some embodiments, nucleic acids encoding CD8a polypeptide encode a mutant CD8a polypeptide. Exemplary nucleotide sequences of CD8a polypeptides are set forth in Table 1. Accordingly, in some embodiments, a nucleic acid sequence encoding a wildtype (WT) CD8a polypeptide is or comprises the nucleic acid sequence of SEQ ID NO: 1.-36- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0125] Nucleic acids of the present disclosure encode, for example, WT or mutant CD8a polypeptides. In some embodiments, mutant CD8a polypeptides exhibit reduced activity (e.g., reduced binding to MHC) relative to the wild-type CD8a polypeptide. In some embodiments, mutant CD8a polypeptides exhibit increased activity (e.g., increased binding to MHC) relative to the wild-type CD8a polypeptide. Exemplary amino acid sequences of CD8a are set forth in Table 2. Accordingly, in some embodiments, a wild-type (WT) CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 27. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 30 or SEQ ID NO: 31. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 34 or SEQ ID NO: 35. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 40 or SEQ ID NO: 41. In some-37- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 42 or SEQ ID NO: 43. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 45. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 46 or SEQ ID NO: 47. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 48 or SEQ ID NO: 49. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 50 or SEQ ID NO: 51. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 59 or SEQ ID NO: 60. In some embodiments, a mutant CD8a polypeptide is or comprises the amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 62.

[0126] Additionally or alternatively, in certain embodiments, a nucleic acid comprises a nucleic acid sequence encoding a mutant CD8a polypeptide (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) and a first reporter or selection marker (e.g., GFP, puromycin resistance). In some such embodiments, the WT or mutant CD8a polypeptide and the first reporter or selection marker are linked by a selfcleaving linker, such as a P2A linker. In certain embodiments, a heterologous WT or mutant CD8a polypeptide and a reporter or selection marker (c.g, GFP, puromycin resistance) are expressed as two separate polypeptides. In some such embodiments, the nucleic acid encoding a WT or mutant CD8a polypeptide and the first reporter or selection marker are separated by a nucleic acid sequence encoding an Internal Ribosomal Entry Site (IRES).

[0127] Additionally or alternatively, in certain embodiments, a nucleic acid comprises a nucleic acid sequence encoding a mutant CD8a polypeptide (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) and a TCR (as-38- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443described elsewhere herein). In some such embodiments, the CD8a and TCR are linked by a self-cleaving linker, such as a P2A linker. In certain embodiments, a heterologous CD8a and a TCR are expressed as two separate polypeptides. In some such embodiments, the nucleic acid sequence encoding a CD8a and a TCR are separated by a nucleic acid sequence encoding an Internal Ribosomal Entry Site (IRES).

[0128] Additionally or alternatively, in some embodiments, a heterologous nucleic acid comprising a nucleic acid sequence encoding a CD8a (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) and / or a TCR of the present disclosure is operably linked to an inducible promoter. In some embodiments, a heterologous nucleic acid comprising a nucleic acid sequence encoding a CD8a gene (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) and / or a TCR of the present disclosure is operably linked to a constitutive promoter.

[0129] Among other things, technologies of the present disclosure provide engineered immune cells (e.g., engineered cytotoxic T cells, engineered CD4+ helper T cells) that comprise a non-endogenous expression vector that includes a nucleic acid sequence encoding a mutant CD8a polypeptide described herein (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a). In some embodiments, engineered immune cells of the present disclosure express mutant CD8a and a TCR (e.g., a native TCR, a non-native TCR, a mimic TCR). In certain embodiments, engineered immune cells can be transduced with a nucleic acid sequence encoding a CD8a polypeptide (e.g., a mutant CD8a described herein) polypeptide. In some embodiments, a coding sequence of a CD8a polypeptide is endogenously present in an engineered immune cell (e.g., engineered cytotoxic -39- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443T cells). In some embodiments, a coding sequence of a WT or mutant CD8a is exogenously provided to an engineered immune cell via a nucleic acid vector, such as a retroviral vector. Technologies of the present disclosure also provide, among other things, methods of using such cells for the treatment of cancer.Vectors

[0130] Many expression vectors are available and known to those of skill in the art and can be used for expression of polypeptides (e.g., TCRs, CD8a polypeptides) provided herein. The choice of expression vector will be influenced by the choice of host expression system. Such selection is well within the level of skill of the skilled artisan. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. Selectable markers include, for example, fluorescent markers (e.g., green fluorescent protein, mCherry, etc.) or antibiotic resistance markers e.g., puromycin resistance, ampicillin resistance, etc.) In some cases, an origin of replication can be used to amplify the copy number of the vector in the cells.

[0131] Vectors also can contain additional nucleotide sequences operably linked to the ligated nucleic acid molecule, such as, for example, an epitope tag such as for localization (e.g., signal sequences, including, for example, ER signaling sequences), e.g., a hexa-his tag or a myc tag, hemagglutinin tag or a tag for purification, for example, a GST fusion, and a sequence for directing protein secretion and / or membrane association.

[0132] Expression of polypeptides, including, for example, T cell Receptors and CD8a polypeptides, can be controlled by any promoter / enhancer known in the art. In some embodiments, a promoter is an inducible promoter, a constitutive promoter, a native promoter (e.g., CD8 or CD4 promoter), or a heterologous promoter. Suitable bacterial promoters are well known in the art and described herein below. Other suitable promoters for mammalian cells, yeast cells and insect cells are well known in the art and some are exemplified below. Selection of the promoter used to direct expression of a heterologous nucleic acid depends on the particular application and is within the level of skill of the skilled artisan. Promoters which can be used include but are not limited to eukaryotic expression vectors containing the SV40 early promoter (Bernoist and Chambon, Nature 290:304-310(1981)), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto etal., Cell 22:787-797(1980)), the herpes thymidine kinase promoter (Wagner etal., Proc. Natl. Acad. Sci. USA 75: 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster etal., Nature -40- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443296:39-42 (1982)); prokaryotic expression vectors such as the P-lactamase promoter (Jay etal., Proc. Natl. Acad. Sci. USA 75:5543 (1981)) or the tac promoter (DeBoer etal., Proc. Natl. Acad. Sci. USA 50:21-25(1983)); see also " Useful Proteins from Recombinant Bacteria": in Scientific American 242:79-94 (1980)); plant expression vectors containing the nopaline synthetase promoter (Herrera- Estrella et al., Nature 505:209-213(1984)) or the cauliflower mosaic virus 35S RNA promoter (Gardner etal., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 510: 1 15-120(1984)); promoter elements from yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions that exhibit tissue specificity and have been used in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., Cell 55:639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409(1986); MacDonald, Hepatology 7:425-515 (1987)); insulin gene control region which is active in pancreatic beta cells (Hanahan etal., Nature 515: 115-122 (1985)), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., Cell 55:647-658 (1984); Adams etal., Nature 515:533-538 (1985); Alexander etal., Mol. Cell Biol. 7: 1436-1444 (1987)), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., Cell 15:485-495 (1986)), albumin gene control region which is active in liver (Pinckert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., Mol. Cell. Biol. 5: 1639-403 (1985)); Hammer et al., Science 255:53-58 (1987)), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al., Genes and Devel. 7:161-171 (1987)), beta globin gene control region which is active in myeloid cells (Magram et al., Nature 515:338-340 (1985)); Kollias et al., Cell 5:89-94 (1986)), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead etal., Cell 15:703-712 (1987)), myosin light chain-2 gene control region which is active in skeletal muscle (Shani, Nature 514:283-286 (1985)), and gonadotrophic releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al., Science 254: 1372- 1378 (1986)).

[0133] In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of an antibody, or antigen binding fragment thereof, in host cells. A typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the polypeptide chains of interest and signals required for efficient polyadenylation of the transcript, ribosome -41- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443binding sites and translation termination. Additional elements of the cassette can include enhancers. In addition, the cassette typically contains a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region can be obtained from the same gene as the promoter sequence or can be obtained from different genes.

[0134] Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase. Alternatively, high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with a nucleic acid sequence encoding a germline antibody chain under the direction of the polyhedron promoter or other strong baculovirus promoter.

[0135] Any methods known to those of skill in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors containing a nucleic acid encoding any of the polypeptides provided herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any site desired can be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers can contain specific chemically synthesized nucleic acids encoding restriction endonuclease recognition sequences.10136] Exemplary plasmid vectors useful to produce the polypeptides provided herein contain a strong promoter, such as the HCMV immediate early enhancer / promoter or the MHC class I promoter, an intron to enhance processing of the transcript, such as the HCMV immediate early gene intron A, and a polyadenylation (poly A) signal, such as the late SV40 poly A signal.

[0137] Genetic modification of engineered immune cells (e.g., T cells) can be accomplished by transducing a substantially homogeneous cell composition with a recombinant DNA or RNA construct. The vector can be a retroviral vector (c.g, gamma retroviral), which is employed for the introduction of the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding a certain TCR (e.g., 1G4 TCR) and / or a certain CD8a polypeptides (c.g, Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, -42- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443Variant 239 CD8a) can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from an alternative internal promoter.

[0138] Non-viral vectors or RNA may be used as well. Random chromosomal integration, or targeted integration (e.g., using a nuclease, transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPRs), or transgene expression (e.g, using a natural or chemically modified RNA) can be used.

[0139] For initial genetic modification of the cells to provide certain TCR (e.g, 1G4 TCR) and / or CD8a polypeptides e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a), a retroviral vector is generally employed for transduction, however any other suitable viral vector or non-viral delivery system can be used. In some embodiments, for example, non-endogenous expression vectors are utilized. Nonlimiting examples of non-endogenous expression vectors include a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector. For subsequent genetic modification of the cells to provide cells comprising an antigen presenting complex comprising at least two co-stimulatory ligands, retroviral gene transfer (transduction) likewise proves effective. 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, etal., Mol. Cell. Biol. 5:431-437 (1985)); PA317 (Miller, etal., Mol. Cell. Biol. 6:2895-2902 (1986)); and CRIP (Danos, etal. Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988)). Non -amphotropic particles are suitable too, e.g., particles pseudotyped with VSVG, RD114 or GALV envelope and any other known in the art.

[0140] Possible methods of transduction also include direct co-culture of the cells with producer cells, e.g., by the method of Bregni, et al., Blood 80: 1418-1422(1992), 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, etal, Exp. Hemat. 22:223-230 (1994); and Hughes, etal., J. Clin. Invest. 89: 1817 (1992).-43- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[01411 Transducing viral vectors can be used to express a co-stimulatory ligand and / or secrete a cytokine (e.g., 4-1BBL and / or IL-12) in an engineered immune cell. In some embodiments, the chosen vector exhibits high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430 (1997); Kido et al., Current Eye Research 15:833-844 (1996); Bloomer etal., Journal of Virology 71:6641-6649, 1997;Naldini etal., Science 272:263 267 (1996); andMiyoshi et al., Proc. Natl. Acad. Sci. U. S. A.94: 10319, (1997)). Other viral vectors that can be used include, for example, adenoviral, lentiviral, and adeno-associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, (1990); Friedman, Science 244: 1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614, (1988); Tolstoshev etal., Current Opinion in Biotechnology 1:55-61(1990); Sharp, The Lancet 337: 1277-1278 (1991); Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322 (1987); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995)). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370 (1990); Anderson etal., U. S. Pat. No. 5,399,346).

[0142] In certain non-limiting embodiments, the vector expressing a presently disclosed TCR (e.g., 1G4 TCR) and / or CD8a polypeptide (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) is a retroviral vector, e.g., an oncoretroviral vector.

[0143] Non-viral approaches can also be employed for the expression of a protein in 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 etal., Proc. Nat'l. 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,-44- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443electroporation, 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). Transient expression may be obtained by RNA electroporation.

[0144] cDNA expression for use in engineered immune cells, as described elsewhere herein, 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.

[0145] The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.Engineered Immune Cells o f the Present Technology

[0146] Technologies of the present disclosure provide, among other things, engineered immune cells (e.g., engineered cytotoxic T cells, engineered CD4+ helper T cells) comprising a CD8a polypeptide (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a, Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) and a T cell receptor that binds to a target antigen (e.g., 1G4 TCR). In certain embodiments, engineered immune cells can be transduced with a vector comprising a nucleic acid sequence that encodes a CD8a polypeptide (e.g., Variant 9 CD8a, Variant 16 CD8a, Variant 17 CD8a, Variant 27 CD8a, Variant 28 CD8a, Variant 32 CD8a, Variant 37 CD8a, Variant 39 CD8a, Variant 54 CD8a, Variant 64 CD8a, Variant 70 CD8a, Variant 84 CD8a, Variant 86 CD8a, Variant 90 CD8a, Variant 98 CD8a, Variant 116 CD8a,-45- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443Variant 123 CD8a, Variant 125 CD8a, Variant 128 CD8a, Variant 137 CD8a, Variant 201 CD8a, Variant 218 CD8a, Variant 227 CD8a, Variant 230 CD8a, Variant 239 CD8a) and / or a vector comprising nucleic acid sequence that encodes a TCR that binds to a target antigen (e.g., 1G4 TCR).[0147[ The presently disclosed subject matter provides, among other things, engineered immune cells and methods of using such cells for the treatment of cancer. The engineered immune cells of the presently disclosed subject matter are T cells.

[0148] The lymphoid lineage, comprising B, T, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Non-limiting examples of immune cells of the lymphoid lineage include T cells, Natural Killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells can be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, T helper 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), Natural killer T cells, and Mucosal associated invariant 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. In certain embodiments, T cells of the presently disclosed subject matter comprise engineered cytotoxic T cells or engineered CD4+ helper T cells.

[0149] In some embodiments, engineered immune cells of the present disclosure comprise an engineered cytotoxic T cell or an engineered CD4+ helper T cell that comprises a TCR that binds to a target antigen and / or a nucleic acid encoding the T cell receptor. In some embodiments, a TCR is a native TCR, a non-native TCR, or a mimic TCR. Examples of mimic TCRs include but are not limited to, TCRm Abs, TCRm BITEs, TCRm CARs, and ImmTACs. In some embodiments, the TCR is 1G4, DMF5, TCR4, MAGE A3, or A6 TCR or an affinity enhanced version thereof (e.g., 1G4 / LY, 1G4 / TS).

[0150] In some embodiments, engineered immune cells of the present disclosure comprises an engineered cytotoxic T cell that lacks detectable expression or activity of a wild-type CD8a polypeptide. In some such embodiments, the wild-type CD8a polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the engineered cytotoxic T cell comprises a deletion, an inversion, a missense mutation, a-46- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443nonsense mutation, or a frameshift mutation in a nucleic acid sequence encoding the wild-type CD8a polypeptide (i.e., wherein the nucleic acid sequence encoding the wild-type CD8a polypeptide is SEQ ID NO: 1) or comprises one or more disruptions in endogenous genes encoding the wild-type CD8a polypeptide (e.g., CRISPR knockouts). By way of example only, one or more endogenous genes may be knocked out using an endonuclease selected from the group consisting of a CRISPR system (e.g., a Cas endonuclease), TALEN, Zinc Finger, transposon-based, ZEN, meganuclease, Mega-TAL, and any combination thereof.

[0151] Gene suppression can be performed in a number of ways. For example, gene expression can be suppressed by knock out, altering a promoter of a gene, and / or by inhibiting transcriptional or translational activity. This can be done at an organism level or at a tissue, organ, and / or cellular level. Gene suppression methods may comprise overexpressing a dominant negative protein. This method can result in overall decreased function of a functional wild-type gene. Additionally, expressing a dominant negative gene can result in a phenotype that is similar to that of a knockout and / or knockdown. Sometimes a stop codon can be inserted or created (e.g., by nucleotide replacement), in one or more genes, which can result in a nonfunctional transcript or protein (sometimes referred to as knockout). For example, if a stop codon is created within the middle of one or more genes, the resulting transcription and / or protein can be truncated, and can be nonfunctional. However, in some cases, truncation can lead to an active (a partially or overly active) protein. If a protein is overly active, this can result in a dominant negative protein.

[0152] In some embodiments, the engineered cytotoxic T cell comprises an inhibitory nucleic acid that specifically targets and inhibits the expression of a nucleic acid sequence encoding the wild-type CD8a polypeptide (i.e., wherein the nucleic acid sequence encoding the wildtype CD8a polypeptide is SEQ ID NO: 1). In some such embodiments, the inhibitory nucleic acid is an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA.

[0153] In some embodiments, engineered immune cells of the present disclosure comprise an engineered cytotoxic T cell that comprises a non-endogenous expression vector that includes a nucleic acid sequence encoding a mutant CD8a polypeptide (i.e., comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62). In some embodiments, the mutant CD8a polypeptide exhibits reduced binding to MHC relative to the wild-type CD8a polypeptide. In some embodiments, the mutant CD8a polypeptide exhibits increased binding to MHC relative to the wild-type CD8a polypeptide.

[0154] In some embodiments, engineered immune cells of the present disclosure comprise an engineered CD4+ helper T cell that comprises a non-endogenous expression vector that -47- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443includes a nucleic acid sequence encoding a CD8a polypeptide (i.e., comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62).

[0155] In some embodiments, engineered immune cells, as described herein, express a heterologous amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 4-53 or 57-62, or a biological equivalent thereof. In further embodiments, a biological equivalent of an amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62 comprises one or more conservative amino acid substitutions relative to SEQ ID NOs: 4-53 or 57-62, respectively. Additionally or alternatively, in some embodiments, the biological equivalent comprises CD8a activity (e.g, binding to MHC) substantially similar or significantly more compared to the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0156] Additionally or alternatively, in some embodiments, the expression levels and / or activity of CD8a polypeptide in an engineered immune cells is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 times higher compared to that observed in a native immune cell, wherein the engineered immune cell is of the same lineage as the native immune cell.

[0157] In certain embodiments, the presently disclosed engineered immune cells (e.g, T cells) expresses from about 1 to about 5, from about 1 to about 4, from about 2 to about 5, from about 2 to about 4, from about 3 to about 5, from about 3 to about 4, from about 4 to about 5, from about 1 to about 2, from about 2 to about 3, from about 3 to about 4, or from about 4 to about 5 vector copy numbers per cell of a CD8a heterologous nucleic acid and / or TCR heterologous nucleic acid.

[0158] In some embodiments, engineered immune cells of the presently disclosed technologies express non-endogenous levels of CD8a (including, e.g., a mutant CD8a) and / or levels of a TCR for the treatment of cancer, e.g., for treatment of tumor. In some such embodiments, engineered immune cells are administered to a subject (e.g., a human subject) in need thereof for the treatment of cancer.

[0159] In some embodiments, the present disclosure provides a composition comprising an effective amount of engineered cytotoxic T cell or engineered CD4+ helper T cells and a pharmaceutically acceptable carrier.

[0160] In some embodiments, technologies of the present disclosure provide a method for mitigating off-target reactivity and / or toxicity in a subject receiving adoptive T cell therapy -48- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443comprising administering to a subject in need thereof, an effective amount of engineered cytotoxic T cells, engineered CD4+ helper T cell, or engineered cytotoxic T cells or engineered CD4+ helper T cell and a pharmaceutically acceptable carrier.

[0161] In some embodiments, engineered immune cells of the present disclosure may further include at least one recombinant or exogenous co-stimulatory ligand. For example, the presently disclosed engineered immune cells can be further transduced with at least one costimulatory ligand, such that the engineered immune cells co-expresses or is induced to coexpress mutant CD8a and / or a TCR (e.g., 1G4 TCR) and the at least one co-stimulatory ligand. Co-stimulatory 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. TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin-alpha (LTa), lymphotoxin-beta O-TP), CD257 / B cell-activating factor (B AFF) / Bly s / THANK / Tall- 1, glucocorticoid-induced TNF Receptor ligand (GITRL), and T F-related apoptosis-inducing ligand (TRAIL), 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). Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, PD-L1 / (B7-H1) that ligands for PD-1. In certain embodiments, the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof. In certain embodiments, the engineered immune cell comprises one recombinant co-stimulatory ligand (e.g., 4-1BBL). In certain embodiments, the engineered immune cell comprises two recombinant co-stimulatory ligands (e.g., 4-1BBL and CD80).

[0162] Furthermore, the presently disclosed engineered immune cells can, in some embodiments, further comprise at least one exogenous cytokine. For example, a presently disclosed engineered immune cell can be further transduced with at least one cytokine, such that the engineered immune cells secrete the at least one cytokine as well as express mutant -49- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443CD8a. In certain embodiments, the at least one cytokine is selected from the group consisting of IL-2, IL- 3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21. In certain embodiments, the cytokine is IL- 12.

[0163] The engineered immune cells (e.g., T cells) can be generated from peripheral donor lymphocytes, e.g., those disclosed in Sadelain, M., etal., Nat Rev Cancer 3:35-45 (2003), in Morgan, R. A. etal. (2006) Science 314: 126-129; in Dupont et al. (2005) Cancer Res 65:5417-5427; Papanicolaou et al. (2003) Blood 102:2498-2505. The engineered immune cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0164] The unpurified source of immune cells can be any known in the art, such as the bone marrow, fetal, neonate or adult or other hematopoietic cell source, e.g., fetal liver, peripheral blood or umbilical cord blood. Various techniques can be employed to separate the cells. For instance, negative selection methods can remove non-immune cell initially. Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and / or stages of differentiation for both positive and negative selections.

[0165] A large proportion of terminally differentiated cells can be initially removed by a relatively crude separation. For example, magnetic bead separations can be used initially to remove large numbers of irrelevant cells. In some embodiments, at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.

[0016] Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.

[0167] Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.

[0168] The cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI). In some embodiments, the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.

[0169] In some embodiments, the engineered immune cells comprise one or more additional modifications. For example, in some embodiments, the engineered immune cells comprise and -50- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443express (is transduced to express) a chimeric co- stimulatory receptor (CCR). CCR is described in Krause etal. (1998) J. Exp. Med. 188(4):619-626, and US20020018783, the contents of which are incorporated by reference in their entireties. CCRs mimic co-stimulatory signals, but unlike, engineered receptors, do not provide a T-cell activation signal, e.g., CCRs lack a CD3ζ polypeptide. CCRs provide co-stimulation, e.g., a CD28-like signal, in the absence of the natural co-stimulatory ligand on the antigen-presenting cell. A combinatorial antigen recognition, i.e., use of a CCR in combination with an engineered receptor, can augment T-cell reactivity against the dual-antigen expressing T cells, thereby improving selective tumor targeting.[0170| In some embodiments, the engineered immune cells are further modified to suppress expression of one or more genes. In some embodiments, the engineered immune cells are further modified via genome editing. Various methods and compositions for targeted cleavage of genomic DNA have been described. Such targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, U. S. Patent Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861;8,586,526; U. S. Patent Publications 20030232410; 20050208489; 20050026157;20050064474; 20060063231; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960, the disclosures of which are incorporated by reference in their entireties. These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration). Cleavage can occur through the use of specific nucleases such as engineered zinc finger nucleases (ZFN), transcription-activator like effector nucleases (TAKEN s), or using the CRISPR / Cas system with an engineered crRNA / tracr RNA ('single guide RNA') to guide specific cleavage. In some embodiments, the engineered immune cells are modified to disrupt or reduce expression of an endogenous T-cell receptor gene (see, e.g., WO 2014153470, which is incorporated by reference in its entirety). In some embodiments, the engineered immune cells are modified to result in disruption or inhibition of PD1, PDL-1 or CTLA-4 (see, e.g. U. S. Patent Publication 20140120622), or other immunosuppressive factors known in the art (Wu et al. (2015) Oncoimmunology 4(7): el016700, Mahoney etal. (2015) Nature Reviews Drug Discovery 14, 561-584).-51- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443Administration

[0171] Engineered immune cells expressing TCRs and / or CD8a polypeptides of the presently disclosed subject matter can be provided systemically or directly to a subject for treating a disease or condition (e.g., cancer). In some embodiments, a subject suffers from or is diagnosed with cancer. In some embodiments, the cancer or tumor is selected from the group consisting of adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.

[0172] In some embodiments, technologies of the present disclosure provide a method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of an engineered cytotoxic T cell, an engineered CD4+ helper T cell, or a composition comprising an effective amount of an engineered cytotoxic T cell or an engineered CD4+ helper T cell and a pharmaceutically acceptable carrier as disclosed herein.

[0173] In certain embodiments, engineered immune cells or compositions comprising engineered immune cells are directly injected into an organ of interest (e.g., a tissue affected by cancer). Additionally or alternatively, engineered immune cells or compositions thereof are provided indirectly to the organ of interest, for example, by administration into the circulatory system or into the tissue of interest. In certain embodiments, engineered immune cells or compositions thereof are injected intratum orally. Expansion and differentiation agents can be provided prior to, during or after administration of cells and compositions to increase production of T cells in vitro or in vivo.

[0174] Engineered immune cells of the presently disclosed subject matter or compositions thereof can be administered in any physiologically acceptable vehicle, systemically or regionally, normally intravascularly, intraperitoneally, intrathecally, or intrapleurally, although they may also be introduced into bone or other convenient site where the cells may find an appropriate site for regeneration and differentiation (e.g., thymus). In certain embodiments, at -52- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443least 1 x 105cells can be administered, eventually reaching 1 x 1010or more. In certain embodiments, at least 1 x 106cells can be administered. A cell population comprising engineered immune cells can comprise a purified population of cells. Those skilled in the art can readily determine the percentage of engineered immune cells in a cell population using various well-known methods, such as fluorescence activated cell sorting (FACS). The ranges of purity in cell populations comprising engineered immune cells can be from about 50% to about 55%, from about 55% to about 60%, about 60% to about 65%, from about 65% to about 70%, from about 70% to about 75%, from about 75% to about 80%, from about 80% to about 85%; from about 85% to about 90%, from about 90% to about 95%, or from about 95 to about 100%. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The engineered immune cells or compositions thereof can be introduced by injection, catheter, or the like. If desired, factors can also be included, including, but not limited to, interleukins, e.g., IL-2, IL-3, IL 6, IL-11, IL-7, IL- 12, IL- 15, IL-21, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, interferons, e.g., y- interferon.[0175| In certain embodiments, compositions of the presently disclosed subject matter comprise pharmaceutical compositions comprising engineered immune cells expressing certain TCRs and / or CD8a polypeptides as described herein with a pharmaceutically acceptable carrier. Administration can be autologous or non-autologous. For example, engineered immune cells expressing TCRs and / or CD8a polypeptides of the present disclosure and compositions comprising the same can be obtained from one subject (e.g., a donor subject), and administered to the same subject or a different, compatible subject (e.g., a recipient subject). Peripheral blood derived T cells of the presently disclosed subject matter or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a pharmaceutical composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising engineered immune cells expressing TCRs and / or CD8 molecules of the present disclosure, it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).Formulations

[0176] Engineered immune cells of the present disclosure and compositions comprising the same can be conveniently provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other -53- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.

[0177] Sterile injectable solutions can be prepared by incorporating the compositions of the presently disclosed subject matter, e.g., a composition comprising engineered immune cells, in the required amount of the appropriate solvent with various amounts of the other ingredients, as desired. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can also be lyophilized. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired. Standard texts, such as “REMINGTON' S PHARMACEUTICAL SCIENCE”, 17th edition, 1985, incorporated herein by reference, may be consulted to prepare suitable preparations, without undue experimentation.

[0178] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. According to the presently disclosed subject matter, however, any vehicle, diluent, or additive used would have to be compatible with the engineered immune cells of the presently disclosed subject matter.

[0179] The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood and lacrimal fluid. The desired isotonicity of the compositions of the presently disclosed subject matter may be accomplished using sodium chloride, or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride is suitable particularly for buffers containing sodium ions.-54- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0180] Viscosity of the compositions, if desired, can be maintained at the selected level using a pharmaceutically acceptable thickening agent. Methylcellulose can be used because it is readily and economically available and is easy to work with. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, and the like. The concentration of the thickener can depend upon the agent selected. The important point is to use an amount that will achieve the selected viscosity. Obviously, the choice of suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, gel or another liquid form, such as a time release form or liquid-filled form).

[0181] Those skilled in the art will recognize that the components of the compositions should be selected to be chemically inert and will not affect the viability or efficacy of the engineered immune cells as described in the presently disclosed subject matter. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard texts or by simple experiments (not involving undue experimentation), from this disclosure and the documents cited herein.

[0182] One consideration concerning the therapeutic use of the engineered immune cells of the presently disclosed subject matter is the quantity of cells necessary to achieve an optimal effect. The quantity of cells to be administered will vary for the subject being treated. In certain embodiments, from about 102to about 1012, from about 103to about 1011, from about 104to about 1010, from about 105to about 109, or from about 106to about 108engineered immune cells of the presently disclosed subject matter are administered to a subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least about 1 x 108, about 2 x 108, about 3 x 108, about 4 x 108, about 5 x 108, about 1 x 109, about 5 x 109, about 1 x 1010, about 5 x 1010, about 1 x 1011, about 5 x 1011, about 1 x 1012or more engineered immune cells of the presently disclosed subject matter are administered to a human subject. The precise determination of what would be considered an effective dose may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art. Generally, engineered immune cells are administered at doses that are nontoxic or tolerable to the patient.

[0183] The skilled artisan can readily determine the amount of cells and optional additives, vehicles, and / or carrier in compositions to be administered in methods of the presently disclosed subject matter. Typically, any additives (in addition to the active cell(s) and / or -55- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443agent(s)) are present in an amount of from about 0.001% to about 50% by weight) solution in phosphate buffered saline, and the active ingredient is present in the order of micrograms to milligrams, such as from about 0.0001 wt % to about 5 wt %, from about 0.0001 wt% to about 1 wt %, from about 0.0001 wt% to about 0.05 wt%, from about 0.001 wt% to about 20 wt %, from about 0.01 wt% to about 10 wt %, or from about 0.05 wt% to about 5 wt %. For any composition to be administered to an animal or human, and for any particular method of administration, toxicity should be determined, such as by determining the lethal dose (LD) and LD50 in a suitable animal model e.g., rodent such as mouse; and, the dosage of the composition(s), concentration of components therein and timing of administering the composition(s), which elicit a suitable response. Such determinations do not require undue experimentation from the knowledge of the skilled artisan, this disclosure and the documents cited herein. And, the time for sequential administrations can be ascertained without undue experimentation.Combination Therapy

[0184] Also provided are methods for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of any of the engineered immune cells provided herein. In some embodiments of the methods disclosed herein, the engineered immune cell(s) are administered systemically, intranasally, intrapleurally, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the subject in need thereof is human.

[0185] Methods for treating cancer may further comprise sequentially, separately, or simultaneously administering to the subject at least one additional therapy selected from among chemotherapy, radiation, and / or immune modulators.

[0186] In some embodiments, immune modulators comprise immune checkpoint modulators. In some embodiments, immune checkpoint modulators comprise, for example, anti-CTLA4 antibodies (e.g., ipilimumab), anti-PD-1 and / or anti-PD-Ll antibodies (e.g., atezolizumab, avelumab, cemiplimab, dostarlimab, durvalumab, nivolumab, pembrolizumab), and / or anti-LAG-3 antibodies (e.g., Relatlimab).

[0187] In some embodiments, immune modulators comprise cytokine-based therapies. In some such embodiments, cytokine-based therapies comprise, for example, aldesleukin, Granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-a2a, interferon-a2b, and / or peginterferon alfa-2b.

[0188] In some embodiments, immune modulators comprise adjuvants. In some such embodiments, adjuvants comprise, for example, imiquimod and / or poly ICLC.-56- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0189] Additionally or alternatively, in some embodiments, methods for treating cancer in a subject in need thereof comprises administering engineered immune cells as described herein to a subject that has received or is receiving radiation therapy, chemotherapy, or a combination of radiation therapy and chemotherapy.

[0190] Methods for treating cancer may further comprise sequentially, separately, or simultaneously administering to the subject at least one additional therapy selected from among bevacizumab, irinotecan hydrochloride, capecitabine, cetuximab, ramucirumab, fluorouracil, ipilimumab, pembrolizumab, leucovorin calcium, trifluridine and tipiracil Hydrochloride, nivolumab, oxaliplatin, panitumumab, regorafenib, and ziv-aflibercept.[01911 In any case, the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.Kits

[0192] In one aspect, the kits of the present technology comprise a therapeutic composition including any of the engineered immune cells disclosed herein in unit dosage form, and / or vectors comprising any of the nucleic acids disclosed herein. In some embodiments, the kit comprises a sterile container which contains therapeutic compositions including the engineered immune cells disclosed herein; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0193] In some embodiments of the kits, the engineered immune cells of the present technology can be provided together with instructions for administering the engineered immune cell to a subject. In some embodiments, the subject is diagnosed with or suffers from cancer. In certain embodiments of the kits, the vectors comprising any of the nucleic acids disclosed herein can be provided together with instructions for using immune cells transduced with said vectors to treat or mitigate any disease or condition described herein. In certain embodiments of the kits, the vectors comprising any of the nucleic acids disclosed herein can be provided together with instructions for transducing CD4+ helper T cells or cytotoxic T cells with expression vector.-57- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443

[0194] The instructions will generally include information about the use of the composition for the treatment of any disease or condition described herein. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment of any disease or condition described herein or symptoms thereof; precautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0195] The at least one engineered immune cell of the present technology may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation (e.g., Kivitz et al., Clin. Ther. 28:1619-29 (2006)).10196] A device capable of delivering the kit components through an administrative route may be included. Examples of such devices include syringes (for parenteral administration) or inhalation devices.

[0197] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of engineered immune cell compositions of the present technology that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and / or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.EXAMPLESExample 1: Materials and Methods

[0198] ELISPOT Assay: IFN-y ELISPOTs were performed using anti-IFN-y-coated plates. T cells and targets were co-cultured at a 5: 1 ratio for 20 hours. Spots were developed with streptavidin-HRP and TMB, then quantified as spot-forming units (SFUs). Real-Time Cytotoxicity: Cytotoxicity was assessed by eGFP loss using Incucyte imaging. CD8+ T cells with CD8a variants were co-cultured with eGFP+ targets, and data were normalized to the initial timepoint. Flow Cytometry: T cells were stained for CD4, CD8a, CD8P, H131 (TCR P-chain), and viability on a MACSQuantl6 to assess CD8a mutation effects on T cell phenotype and viability. Peptide Avidity Assay: T cells were stimulated with peptide concentration gradient and functional responses (TNFa, IFNy, IL2, CD107a) were measured by flow cytometry. Sequencing and Data Analysis: Genomic DNA was extracted post--58- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443enrichment, and CD8a CDR regions were PCR-amplified and sequenced by NGS. Enriched mutations were analyzed for frequency across rounds to identify potent CDR mutations.Example 2: Functional Characteristics of Mutant CD8 Co-receptors of the Present Technology

[0199] We demonstrated using CRISPR / Cas9 that altering the CD8 co-receptor by generating CD8a homodimers in CD8 T cells transduced with the 1G4 TCR significantly reduces off-target reactivity. However, this modification also dampens the potency of the TCR, particularly at low effector-to-target ratios (FIGs. 2A-2F). Thus, CD8aa improves the safety profile of affinity-enhanced TCRs while maintaining on-target reactivity at certain E: T ratios.

[0200] Given the potential safety advantages of native TCRs, we aimed to enhance efficacy by modifying the CD8 co-receptor. To this end, we generated a saturation mutagenesis library targeting the CDR regions of CD8 involved in MHC binding (FIGs. 4A-4C). After multiple rounds of enrichment using a Jurkat NF AT reporter cell line transduced with the CD8a mutant library and the native 1G4 TCR, we identified several mutations that significantly enhance the potency of the native TCR (FIGs. 3A-3E, FIG. 7, FIGs. 8A-8B, FIG. 10, FIG. 11). The CD8a mutants enhance TCR signaling and cytotoxicity in a TCR / HLA-independent manner. See FIGs. 5A-5F. Further, CD8a mutants do not increase off-target reactivity that was previously identified with affinity enhanced 1G4 TCR (FIG. 6), supporting their potential for improving TCR-based therapies. Increased CD8[3 surface expression in select mutants suggests enhanced a[3 heterodimer stability or retention. See FIG. 12.

[0201] Currently, we are validating these mutations through additional T cell functional assays such as cytotoxicity and IFN-G ELISPOT to ensure that the enhanced potency does not compromise the natural safety mechanisms of the native TCR. We are also testing these CD8-TCR combinations with other HLA types beyond HLA-A02, given that CD8a interacts with the most conserved regions of MHC class I molecules, which could expand the therapeutic applicability across different patients.Example 3: Materials and Methods

[0202] Cell lines and culture: T2 (174xCEM. T2; ATCC® CRL-1992™). Maintained in IMDM (Gibco) supplemented with 10% FBS (GeminiBio), 1% penicillin / streptomycin (P / S; Gibco), 1% L-glutamine. The retroviral packaging line 293GP (gag-pol; gift of C. Klebanoff) and lentiviral producer cell line Lenti-X (Takara) was cultured in DMEM + 10% FBS + Pen-Strep. Jurkat cells (NF AT reporter and parental) were maintained in RPMI-1640 (Gibco) + 10% FBS + Pen-Strep + 1% glutamine.

[0203] Human melanoma lines A375 and SK -MEL-37 were cultured in DMEM + 10% FBS + Pen-Strep + 1% glutamine. HCC70 isogenic lines (mutant PIK3CA; gift of C. Klebanoff) and -59- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443COS-7 stably expressing HLA-A*03:01 (gift of C. Klebanoff) were maintained in DMEM + 10% FBS +P / S.

[0204] Mycoplasma & authentication: All lines were tested mycoplasma-negative using the Lonza MycoAlert™ Mycoplasma Detection Kit.[0205 { Primary human T cells: Donors and PBMC isolation. Whole blood from healthy donors was obtained under IRB -approved protocols with written informed consent. PBMCs were isolated by density-gradient centrifugation using Lymphocyte Separation Medium (Corning). CD8+T-cell isolation and culture. CD8+T cells were purified by negative selection (STEMCELL Technologies) and stimulated with ImmunoCult™ (STEMCELL) in ImmunoCult-XF medium.

[0206] Plasmid and Peptides

[0207] TCRs and CD8 construct: The 1G4 and TCR4 TCR plasmid were gifts from C. Klebanoff. MAGE-A3 cDNA was synthesized (GenScript). Human CD8a / p ectodomain and full-length constructs were synthesized (GenScript). Site-directed mutagenesis was performed using [NEB Q5 / Agilent] per manufacturer’s instructions; primers were designed with the NEB SDM tool.

[0208] Peptides. Peptides (>95% purity; GenScript) were dissolved at 20 mg / mL in 100% DMSO and stored at -80 °C.

[0209] Jurkat NFAT-eGFP reporter line and CD8a library: Reporter line. A lentiviral NFAT-eGFP reporter (BPS Biosciences / catalog #79922) was used to transduce Jurkat cells. Single cells were sorted by FACS, expanded, and the highest signal -to-noise clone was selected for screening.

[0210] CD8a library. A saturation mutagenesis library targeting CD8a CDR / loop residues implicated in MHC-I binding (GenScript) was packaged in HEK-293GP (see below). Jurkat reporter cells were transduced in non-TC-treated 6-well plates with viral supernatant + 6 pg / mL polybrene; spinoculation at 32 °C, l,000xg for 1 h. MOI < 0.3 was used to favor single plasmid / cell integration. Genomic DNA was extracted (Qiagen), the CD8a region PCR-amplified, and amplicon-seq performed by Illumina sequencing. Library representation was confirmed to be 100%.

[0211] Library screening (Jurkat NFAT-eGFP): Jurkat library cells were co-cultured overnight with T2-A*02:01 cells pulsed with NY-ESO-1 peptide (or unpulsed controls). Cells were washed (FACS buffer), stained with DAPI, and the top 5% eGFPhighlive singlets were sorted and expanded. After each round, half the cells were harvested for amplicon-seq of CD8a; the remainder underwent the next enrichment. Three rounds were performed.-60- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[0212| Viral transduction: Retroviral TCR transduction. 293GP were plated on poly-D-lysine-coated 60-mm plates at 1.6><106cells / plate. Cells were transfected with 6 pg pMSGVl-TCR + 3 pg RD114 envelope using Lipofectamine 3000 (Invitrogen). Viral supernatant was collected at 48 h, loaded onto Retronectin-coated (10 pg / mL; Takara) non-TC-treated 24-well plates, and centrifuged at 2,000*g, 32 °C, 2 h. Stimulated T cells (l-5><105 / well) were added and spinoculated at 1,500 rpm, 15 min. Transduction was assessed at day 3-4 by surface mTCR staining. Lentiviral production (general). Lenti-X were transfected with Lipofectamine3000. Supernatants harvested at 48-72 h, filtered (0.45 pm), and used fresh for transduction.[0213| Cytotoxicity (live-cell imaging): Real-time killing (IncuCyte S3): Tumor cells expressing GFP-NLS were plated in 96-well plates (Coming 3903) at 4xl04cells / well (100 pL RPMI) and incubated 24 h. Effector lymphocytes were added at indicated E: T ratios, plates rested 20 min at RT, then imaged on IncuCyte S3. Analysis used IncuCyte Basic Analysis.[0214| Alignment of HLA: Full-length HLA-I sequences were downloaded from IPD-IMGT / HLA (version / date). Sequences were aligned using Clustal Omega (EMBL-EBI) with default parameters; only full-length alleles were included in downstream analyses. Data were analyzed and visualized in Python using Matplotlib (v3.8.2) and Seaborn (vO.13.2).

[0215] Flow Cytometry: Cells were washed in FACS buffer (PBS, 2% FBS, 2 mM EDTA) and stained 30 min on ice, protected from light. Antibodies included anti-CD3 FITC (eBioscience #11- 0037-42) stained at 1:500. Anti-CD4 FITC (eBioscience #11-0048-42) stained at 1:500. Anti-CD8 APC (BioLegend #344722) stained at 1:500. APC conjugated NY-ESO-1 / A02 tetramer provided by the NIH or MAGE-A3 / A01 tetramer were purchased, stained at 1:1000 to 1:2000. Anti-Vbl3.1 FITC clone H131 stained at 1:50 (Biolegend #362404). CD8a (HIT8a or SKI), CD8P (clone SID8BEE and clone 2ST.85H7), Zombie NIR (BioLegend) for live / dead, and NY-ESO-1 / A*02:01 tetramers (APC, Miltenyi Biotec, catalog #130-135-299) or MAGE-A3 / A*01:01 tetramer (APC, Creative Biolabs, Cat#MHC-LC126). Anti-Vpi3.1 H131 (FITC; BioLegend #362404) was used where indicated. Data were acquired on a MacsQuantl6 and analyzed in FlowJo vlO. Samples are washed and analyzed with MacsQuantl6 flow cytometer.10216] ELISpot Assay: PVDF plates (Multi Screen-IP, Millipore #S2EM004M99) were prewetted with 70% ethanol, coated with anti-IFN-y 1-DIK (Mabtech #3420-3-1000; 10 pg / mL, 100 pL / well, 4 °C overnight), washed, and blocked (complete RPMI, 2 h, 37 °C). T cells (5x 04 / well) were co-cultured with peptide-pulsed targets (20 pg / mL, 2 h pulse, washed; lx 04 / well) >20 h at 37 °C. Plates were washed (PBS / 0.05% Tween-20), incubated with -61- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443biotin-anti-IFN-Y 7-B6-1 (Mabtech #3420-6-250; 2 pg / mL, 2 h, 37 °C), washed, developed with Vectastain Elite ABC HRP (Vector #PK-6100, 1 h, RT), then AEC substrate (see original recipe), stopped with water, dried, and counted.

[0217] CRISPR / Cas9 Gene Knockout in Primary T cells: Stimulated T cells were washed in PBS. 10-20x10® T cells were mixed with 80pMol modified sgRNA (Synthego) and lOOpMol CleanCap Cas9 protein (Synthego) and electroporated in cuvettes using the Lonza electroporater (cells resuspend in P3 buffer, recommended program was used. sgRNA sequences: CD8A: CGCCAGGCCGAGCCAGTTCC (SEQ ID NO: 54); CD8B:TCAGUAACAUGCGCAUCUAC (SEQ ID NO: 55). Cells were immediately transferred to pre-warmed ImmuncultX media + 300 IU IL-2. 5 days after electroporation cells were isolated for CD8aa or CD8KO pure population by negative selection using Miltenyi beads for CD8 or CD8p.

[0218] Animal Studies: All animal procedures were performed in accordance with an MSKCC Institutional Animal Care and Use Committee-approved protocol. Four to six-week- old female NOD. C^-Prkdc^ Il2r^mlw}lISzi (NSG) mice were purchased from Jackson Laboratory and housed in pathogen-free conditions at the MSKCC vivarium. The mouse room maintained a 12-hour light / dark cycle, temperature of 65-75 “F and humidity levels of 40- 60%. Threex106cell s of the indicated I ICC70 isogenic cell lines (I ICC70-WT PIK3CA or HCC70-Mut 7X3G4) were subcutaneously implanted into the right flank of mice. Mice were randomized to indicated treatment groups once tumors were established to ~75 mm3. Tumor width and diameter as well as animal weights were measured by an investigator blinded to treatment conditions at baseline and twice weekly thereafter. TCR4-tranduced CD8+T cells were intravenously transferred at day 11 (3 * 106). Control groups received an equal number of CD8" T ceils transduced with a previously described HLA-A*03:01-restricted Flu-specific TCR or PBS. The maximum tumor size permitted by the approved protocol is 2,000 mm3and this value was not exceeded.

[0219] Statistics and reproducibility: No statistical methods were used to predetermine sample size. Appropriate statistical tests were used to analyze data, as described in each figure legend. Statistical analyses were performed with GraphPad Prism version 8.4 software.Significance was preset at P < 0.05. In vitro experimental data were generated from two or more independent experiments containing n = 3 biological replicates per condition per experiment. For in vivo mouse experiments, treatment groups had n = 5 mice per condition, and control groups had n = 5 mice. Mice were randomized into three groups after tumor implantation. An investigator blinded to treatment groups performed both data acquisition and -62- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443data analysis. For both in vitro and in vivo experiments, no data were excluded from any analysis.

[0220] Survival of tumor-bearing mice was analyzed using Kaplan-Meier survival curves generated in GraphPad Prism (version 10.6.1). Statistical significance between groups was assessed using the Log-rank (Mantel-Cox) test, and when indicated, the Gehan-Breslow-Wilcoxon test was used to account for early events. Median survival and 95 % confidence intervals were reported. Pairwise comparisons were performed between relevant groups, and p < 0.05 was considered statistically significant.Example 4: Enhancing Antigen-specific T cell Activation by Use of CD8a Mutant Forms

[0221] One approach for improving native TCR activity to levels necessary for consistently effective clinical use is to overexpress CD8 together with the TCR19. However, overexpression of CD8a or CD8aP wild type molecules in T cells containing low affinity TCRs has consistently failed to increase cytotoxic activity, suggesting limited benefit to simple CD8 enhancement with TCRs of low affinity to peptide / HLA (KD ~15 pM to -200 pM20,21). We expanded this previous observation, by testing the co-expression of the CD8a homodimer or the CD8aP heterodimer to three additional tumor-specific TCRs (FIGs. 13A-13B, FIG. 17A).We demonstrated in in vitro tumor killing assays no benefit to adding CD8a or CD8aP to isolated CD8+T cells expressing the native TCR 1G4 / TS reactive with the NY-ESO-1 peptide / HLA-A*02:0122complex, the native TCR4to mutant PIK3CA (H1047L) peptide / HLA-A*03:01 complex23, or the native TCR to MAGE- A3 peptide / HLA-A*01:01 complex24(FIGs. 13A-13B, FIGs. 17B-17H). The clinically evaluated affinity enhanced TCRs 1G4 / LY (NY-ESO-1) and a3a (MAGE) showed greater potency than their native counterparts when coexpressed with CD8a or CD8aP (FIG. 13B; FIGs. 17B-17C, 17G-17H)25. Based on this, to identify CD8 molecules that enhance native TCR activity, we selected the native NY-ESO-1 -specific TCR 1G4 / TS as the model, based on its suboptimal functional avidity. We performed a CD8a saturation mutagenesis screen using a library that altered residues either within or adjacent to the CD8a CDR regions previously implicated in pHLA binding (FIGs. 3A, 13C)26.Every amino acid from CD8a position L46-S52 (CDR1), S74-N76 (CDR2) andN120-S121(CDR3) were changed to all 20 standard amino acids. The library, together with the native 1G4 / TS TCR, as multi ci str onic constructs, was then introduced into a JurkatNFAT-eGFP reporter cell line to enable identification of TCR activated cells based on GFP expression (FIG. 18A). Cells were successfully transduced at a MOI < 0.3 with all 228 mutated CD8a sequences and enriched by MACS for the VP chain of the 1G4 TCR (FIGs. 18B-18C).-63- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[02221 Upon co-culture with HLA-A*02:01+T2 cells pulsed with NY-ESO-1 peptide, the top ~5% signaling GFP-positive cells were isolated by FACS sorting to identify variants resulting in the highest level of TCR activation (FIG. 3C). As a control, library expressing cells were co-cultured with unpulsed T2 cells. Each enriched, positive sorted cell population was cultured and resorted 2 more times (FIG. 13D). Higher GFP+signal was observed after each sorting round when library cells were co-cultured with T2 cells pulsed with NY-ESO-1 overnight (FIG. 13E). We observed consistent enrichment of several CD8a variants across the three screens (FIG. 3D). Enriched CD8a variants were sequenced after each of the three independent sorting rounds and ranked based on their fold-changes (FIG. 18D). Overall, seventeen CD8a mutations showed significant enrichment (log2 fold-change >2, <0.05) relative to the baseline library across the screens (FIG. 13F).Example 5: Mutant CD8a Variants Enhanced T cell Cytotoxicity across Multiple TCR-HLA Allelic Contexts[0223 | To validate and characterize the top 17 CD8a mutations identified as potential hits to enhance T cell activation in our screen, we engineered primary isolated CD8+T cells expressing the native TCR 1G4 / TS that also expressed either wild-type CD8aP or CD8aP with a selected mutation. The introduction of CD8P for in vitro cytotoxicity assays is necessary to provide physiological signaling and maximize cytotoxicity. Live-cell imaging of cancer cell killing using the melanoma cell line A375 (NY-ESO-U, HLA-A*02:01+) revealed that the four CD8a mutations (L46S, L46T, L47Q, and S121N) consistently enhanced tumor cell killing across multiple different T-cell donors (FIGs. 14A-14D). A comprehensive overview of all tested CD8 mutants across all donors is shown (FIGs. 19A-19F).[0224| For a TCR enhancement strategy to be broadly useful, it should ideally augment TCR CD8 potency across different HLA-I alleles and epitope classes (cancer-germline antigen versus neoantigen). To evaluate whether the identified CD8-enhancing mutations function across a diverse range of HLA-I alleles beyond HLA-A*02:01, we aligned all 5,184 full-length HLA-A allele sequences available in the IPD-IMGT / HLA database using Clustal Omega (vl.2.4) and mapped amino acid variability across structural domains (FIG. 14E)27. All of the selected CD8a mutations were predicted to interact with HLA a3 domain or P2 microglobulin (P2M), a region which exhibited minimal sequence variability across all HLA-A types (FIG.14F). Based on this analysis, we hypothesized that mutations in CD8a would function broadly across multiple HLA-I alleles, in contrast to the strict peptide / HLA specificity observed in TCR-based recognition. We aligned 6375 sequences for HLA-B and 4268 sequences for HLA-C and found that the a3 domains of HLA-B and HLA-C are among the least polymorphic -64- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443(FIGs. 20A-20D). Based on this, we hypothesized that a CD8 variant enhancing TCR signaling should also work for multiple HLA-B or HLA-C restricted TCRs.

[0225] To test whether the identified CD8 variants work with multiple HLA-A restricted TCRs, we evaluated the top-performing CD 8a mutants in distinct TCR-HLA contexts representative of the most common HLA-A alleles in human populations28. Specifically, we tested 7) a native HLA-A*03:01 restricted TCR (TCR4) targeting a shared neoantigen derived from the recurrent PIK3CA (H1047L) hotspot mutation complex23, and 2) an HLA-A*01:01 restricted native TCR targeting the cancer-germline antigen MAGE-A3 complex24. Live-cell imaging of the HLA-A*03:01+breast adenocarcinoma cell line HCC70 expressing the PIK3CA (H1047L) mutation validated the enhanced antitumor potency of isolated CD8+T cells coexpressing TCR4 plus the top CD8 variants compared with T cells transduced either with TCR4 alone or in combination with wild-type CD8 (FIG. 14G). Similarly, isolated CD8+T cells co-expressing MAGE A3 TCR plus the same CD8 variants demonstrated enhanced cytolytic activity against A375 cells which naturally co-express HLA-A*01:01 and MAGE-A3 relative to the co-expression of wild-type CD8 (FIG. 14H). Together, these results confirm that co-expression of screen identified CD8 variants can enhance the in vitro antitumor potency of multiple TCRs and prevalent restricting HLA-I alleles, supporting their potential for broad applicability.Example 6: Retention of TCR Specificity with CD8 Mutant Enhancement

[0226] In affinity enhanced TCRs, increased affinity to pHLA often exhibit increased off-target reactivity15,29. In contrast, native TCRs are naturally occurring TCRs shaped by thymic selection, typically resulting in minimal reactivity against normal tissue antigens30,31. To evaluate whether the increased potency conferred by our validated CD8a variants comes at the cost of undesired off-target activation of native TCR expressing T cells, we co-cultured 1G4 / TS TCR transduced isolated CD8+T cells which co-express a CD8 variant against HLA-A*02:01+target cells presenting NY-ESO-1 and known off-target peptide sequences29. As controls, target cells were co-cultured either with 1G4 / TS TCR T cells co-transduced with CD8 wild-type or T cells transduced with the affinity-enhanced 1G4 / LY TCR. We previously identified eight human proteome derived off-target peptides for the affinity enhanced TCR 1G4 / LY using PresentER, a cell-based genetic screening platform29,32. The off-target peptide sequences typically differed from the NY-ESO-1 sequence, SLLMQWITQC (SEQ ID NO: 56), by as many as 5-7 amino acids. While the affinity enhanced 1G4 / LY TCR recognized the 8 off-target peptides, the addition of the top performing CD8a mutants, L46S, L46T, L47Q, and S121N, co-expressed with CD8[3 and the native 1G4 / TS TCR allowed it to retain the same -65- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443reactivity profile as the 1G4 / TS TCR alone (FIG. 15A). Therefore, potency could be increased by CD8 mutation without altering off-target reactivity.

[0227] We confirmed the influence of the two top CD8 variants on TCR specificity using two additional model systems. First, we explored the cross-reactivity profile of the native MAGE-A3 TCR restricted by HLA-A*01:01. As a control, we used the a3a affinity-enhanced variant of this TCR which caused lethal toxicities resulting from off-target recognition of a peptide derived from Titin, a protein highly expressed by cardiac myocytes15,16. In this experiment, we transduced HLA-A*01:01 into T2 cells knocked out for the HLA-A *02:01 allele. After modification with the CD8 mutants, isolated CD8+T cells expressing the native MAGE-A3 TCR did not show reactivity to Titin by IFNy ELISpot (FIG. 15B). By contrast, the a3a affinity-enhanced TCR showed strong reactivity to this off-target peptide. Second, we tested whether the CD8 variants altered the cross-reactivity profile of the native TCR4 which recognizes the PIK3CA (H1047L) neoantigen presented by HLA-A*03:01. Using alanine / glycine scanning, we compared TCR4’s recognition motif when expressed by isolated CD8+alone or in combination with either the L46S or S121N CD8 variants. We discovered that the CD8a modified variants retained the exact same reactivity profile as the native TCR4 T cells alone (FIG. 15C).

[0228] Taken together, these data show that the mutations in CD8 which enhance cytotoxicity maintain the cross-reactivity profile of the native TCR across multiple receptors and restricting HLA alleles. Thus, the strategy of modifying the CD8 while retaining the native TCR structure can both improve potency and retain specificity across multiple HLA-A alleles and different epitope targets in several clinically relevant systems.Example 7: Structural insights into TCR Function Enhancing CD8a Variants

[0229] We hypothesized that the improved cytotoxic function observed in T cells expressing one of the CD8 variants and a native TCR was due to increased binding affinity of the CD8 mutants to invariant regions of HLA-I molecules. We examined the structural basis underlying the functional enhancements observed with our identified CD8a variants using the crystal structure of CD8aa in complex with HLA-A*02 (PDB: 1AKJ)26. In the wild-type model, the hydrophobic CDR1 residue L46 sits in a highly polar interfacial environment formed by the residues of P-2M K58, HLA A02 D122 and CD8a Q44 / R25 (FIG. 15D, upper panels).Mutation L46S introduces a polar hydroxyl group, which allows potential hydrogen bond formation and reduces steric hindrance, thereby enhancing local complementarity and stability of the CD8-HLA interface. In the CDR3 loop of CD8a, S121 forms a hydrogen bond with the HLA backbone (FIG. 15D, lower panels). Mutation S121N introduces a longer side chain -66- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443containing an amide group position to interact with residues Q226 and D227, creating dual hydrogen-bond capacity that might further stabilize the interface.Example 8: CD8 Mutants Promote Increased Antigen Dependent TCR Signaling

[0230] To understand possible causes for the enhanced functionality of the CD8a mutants, we performed additional p / HLA multimer binding and functional activation assays. Isolated CD8+T cells expressing one of the CD8 mutants promoted increased, albeit modest, p / HLA multimer binding to both the 1G4 and MAGE- A3 TCRs compared with T cells expressing wild-type CD8 in two donors (FIG. 21). To quantify the functional enhancement conferred by this binding increase by CD8 mutants, we expressed 1G4 / TS TCR with either wild-type CD8 or CD8a mutants in the Jurkat NF AT reporter system (FIG. 15E). Importantly, introduction of the mutant did not result in any tonic signaling (FIG. 15E, first panel). Peptide dose-response curves revealed significant leftward shifts in the half-maximal effective peptide concentration (ECso) values, indicating enhanced sensitivity to antigen stimulation (FIG. 15F). Wild-type CD8 required 1.00 pM peptide concentration for half-maximal activation, while L46S and S121N mutants achieved EC50 values of 0.66 pM and 0.38 pM, respectively (FIG. 15G). This represents a 1.5-fold and 2.6-fold improvement in peptide sensitivity for L46S and S121N variants, respectively, demonstrating quantifiable potency enhancement in TCR activation. Notably, ECso values of ~lpM are within the physiological range of peptide concentrations expected to activate T cells in vivo, underscoring the clinical relevance of these enhancements 33,3410231] All together, these data provide mechanistic insight, as structural modeling suggests, that CD8 mutations enhance interactions with HLA leading to an increased structural avidity. This enhancement translates into improved functional TCR activation thresholds and cytotoxicity in vitro at physiologically relevant peptide concentrations.Example 9: CD8 Variants Augment TCR Therapeutic Potency In vivo

[0232] We next tested the potential of the CD8a variants to enhance therapeutic function of a TCR in vivo using the PIK3CA (H1047L) public neoantigen model. Three million HCC70 cells (an HLA-A*03:01+breast adenocarcinoma cell line harboring the mutant PIK3CA (H1047L)) were engrafted into immune-deficient NOD scid gamma (NSG) mice. We tested whether the adoptive cell transfer of the top two CD8 variants, L46S and S121N, in combination with TCR4 transduced into isolated CD8+T cells improves the therapeutic effect compared to wildtype CD8. T cells transduced with an HLA-A*03:01 -restricted influenza nucleoprotein (Flu) TCR served as a negative control.-67- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443[02331 Groups of mice bearing 100mm3tumors were assigned to receive by treatment i.v. by three million cells of either: (1) TCR4 CD8 wild-type transduced isolated CD8+T cells, (2) TCR4 CD8 variant L46S transduced CD8+T cells, (3) TCR4-CD8 variant S121N transduced CD8+T cells, (4) Flu-specific TCR transduced CD8+T cells, or (5) PBS control (FIGs. 16A- 16B) As a stress test, no supportive cytokines were given after adoptive transfer. Mice transduced with TCR4 CD8 L46S variant and TCR4 CD8 121N variant exhibited reduced tumor growth compared to TCR4 CD8 wild-type transduced CD8+T cells (FIG. 16C). The S121N variant significantly decreased tumor volume (p = 0.03, unpaired two-tailed t-test), while the L46S variant showed a similar trend but did not reach statistical significance (p=0.08). Kaplan-Meier analysis demonstrated a significant survival benefit in mice receiving TCR4 T cells co-expressing engineered CD8 variants compared with the WT CD8aP construct. The L46S mutation increased median survival from 29 to 36 days (Log-rank test, p = 0.011), and the S121N mutation produced a similar effect (p = 0.0086). (FIG. 16D). This was not due to adverse effects of the transferred cells or CD8 mutants as monitored by animal weight which stayed comparable (FIG. 16E). Hence, the CD8 variants L46S and S121N improved the therapeutic efficacy of the engineered TCR-T cells in vivo. These data indicate that selective alteration of the CD8 co-receptor can significantly enhance the in vivo efficacy of TCR4 transduced T cells.Example 10: CD8aa Decreases Off-tarset Reactivities of an Affinity-enhanced TCR while Maintaining On-tar et Reactivity

[0234] As a further proof of the concept for this strategy, we postulated that because increasing CD8 affinity for HLA enhanced the function of native TCRs, then the converse action of reducing CD8 interactions with HLA might improve the specificity of affinity-enhanced TCRs. Such affinity enhanced TCR have sufficient potency, but lack on-target selectivity. These latter TCRs (with affinities <3 pM) typically function independently of the CD8 co-receptor35. To test whether modulating the CD8 co-receptor of affinity-enhanced TCR-expressing T cells could reduce off-target reactivity without compromising on-target function we used CRISPR / Cas9 in primary human isolated CD8+T cells to generate T cells lacking either CD8a (CD8- / -) or CD8[3 (CD8aa) and transduced them with the clinically used affinity-enhanced 1G4 / LY TCR specific for the NY-ESO-1 epitope (FIG. 22A)12,36. MACS separation enriched CD8aa and CD8- / - populations and flow cytometry confirmed comparable TCR expression (FIG. 22B)

[0235] IFNy secretion, cytotoxicity, ELISpot assays, tetramer staining, and CD8 expression were examined. Both CD8aa and CD8KO 1G4LY T cells showed reduced IFNy secretion -68- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443patterns resembling those of native 1G4TS T cells while CD8WT enhanced 1G4LY T cells produced robust IFNy responses to multiple off-target peptides (FIG.22C). For killing of A375, CD8aa and CD8WT 1G4LY T cells achieved comparable cytotoxicity, while CD8K0 cells were less effective (FIG.22D). In A375 and SKMEL37 tumor co-cultures, CD8a overexpression failed to enhance cytotoxicity with tumor growth suppression comparable to parental CD8 wild type or CD8aa T cells (FIG.23A). CD8a overexpression increased MHC binding independent of TCR recognition (FIGs.23B-23C). These findings illustrate a trade-off between improved specificity and reduced cytotoxic sensitivity and underscore the potential of CD8aa to act as a safety-enhancing co-receptor variant for affinity-enhanced TCRs, still capable of effectively killing cancer cells.

[0236] Exemplary CD8 receptor sequences utilized herein are shown in Tables 1 and 2.Table 1: Exemplary CD8a nucleic acid sequencesSEQ ID NO: Nucleic acid sequenceSEQ ID NO: 1 ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCT (CD8a WT) TGCTGCTCCACGCCGCCAGGCCGAGCCAGTTCCGGGTGT CGCCGCTGGATCGGACCTGGAACCTGGGCGAGACAGTG GAGCTGAAGTGCCAGGTGCTGCTGTCCAACCCGACGTCG GGCTGCTCGTGGCTCTTCCAGCCGCGCGGCGCCGCCGCC AGTCCCACCTTCCTCCTATACCTCTCCCAAAACAAGCCCA AGGCGGCCGAGGGGCTGGACACCCAGCGGTTCTCGGGC AAGAGGTTGGGGGACACCTTCGTCCTCACCCTGAGCGAC TTCCGCCGAGAGAACGAGGGCTACTATTTCTGCTCGGCC CTGAGCAACTCCATCATGTACTTCAGCCACTTCGTGCCG GTCTTCCTGCCAGCGAAGCCCACCACGACGCCAGCGCCG CGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCC CTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGG GGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTC CTTCTCCTGTCACTGGTTATCACCCTTTACTGCAACCACA GGAACCGAAGACGTGTTTGCAAATGTCCCCGGCCTGTGG TCAAATCGGGAGACAAGCCCAGCCTTTCGGCGAGATACG TCTAATable 2: Exemplary CD8a amino acid sequences-69- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443SEQ ID NO: Amino Acid SequenceSEQ ID NO: 2 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a WT) KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLP AKPTTTP APRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 3 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG (CD8a WT) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS DFRRENEGYYFCS AL SN SIM YFSHFVPVFLP AKPTTTP APRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 4 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 9) KCQVHLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA L46H EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 5 SQFRVSPLDRTWNLGETVELKCQVHLSNPTSGCSWLFQPR (CD 8 a variant 9) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL L46H SDFRRENEGYYFC S ALSNSIMYF SHF VP VFLP AKPTTTP APR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 6 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 16) KCQVSLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE L46S GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLP AKPTTTP APRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 7 SQFRVSPLDRTWNLGETVELKCQVSLSNPTSGCSWLFQPRG (CD 8 a variant 16) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS L46S DFRRENEGYYFCSALSNSIMYFSHFVPVFLP AKPTTTP APRP-70- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 8 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 17) KCQVTLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE L46T GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 9 SQFRVSPLDRTWNLGETVELKCQVTLSNPTSGCSWLFQPRG (CD8a variant 17) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS L46T DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 10 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 27) KCQVLQSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA L47Q EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPWKSGDKPSLSARYV SEQ ID NO: 11 SQFRVSPLDRTWNLGETVELKCQVLQSNPTSGCSWLFQPR (CD8a variant 27) GAAASPTFLLYL SQNKPKAAEGLDTQRF SGKRLGDTFVLTL L47Q SDFRRENEG YYFC S ALSNSIMYF SHF VP VFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 12 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 28) KCQVLGSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA L47G EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPWKSGDKPSLSARYV4915-4673-0127.1Atty. Dkt. No.: 115872-3443SEQ IDNO: 13 SQFRVSPLDRTWNLGETVELKCQVLGSNPTSGCSWLFQPR (CD8a variant 28) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL L47G SDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 14 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 32) KCQVLKSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA L47K EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPWKSGDKPSLSARYV SEQ IDNO: 15 SQFRVSPLDRTWNLGETVELKCQVLKSNPTSGCSWLFQPR (CD 8 a variant 32) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL L47K SDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGD KPSLSARYV SEQ IDNO: 16 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 37) KCQVLTSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE L47T GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ IDNO: 17 SQFRVSPLDRTWNLGETVELKCQVLTSNPTSGCSWLFQPRG (CD8a variant 37) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS L47T DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ IDNO: 18 M ALP VTALLLPL ALLLEI AARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 39) KCQVLYSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE L47Y GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP-72- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ IDNO: 19 SQFRVSPLDRTWNLGETVELKCQVLYSNPTSGCSWLFQPR (CD 8 a variant 39) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL L47Y SDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 20 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 54) KCQVLLFNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE S48F GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ IDNO: 21 SQFRVSPLDRTWNLGETVELKCQVLLFNPTSGCSWLFQPRG (CD 8 a variant 54) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS S48F DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 22 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 64) KCQVLLSDPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE N49D GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 23 SQFRVSPLDRTWNLGETVELKCQVLLSDPTSGCSWLFQPRG (CD 8 a variant 64) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS N49D DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 24 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 70) KCQVLLSIPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE-73- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443N49I GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 25 SQFRVSPLDRTWNLGETVELKCQVLLSIPTSGCSWLFQPRG (CD 8 a variant 70) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS N49I DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKP SLSARYV SEQ ID NO: 26 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 84) KCQVLLSNDTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA P50D EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 27 SQFRVSPLDRTWNLGETVELKCQVLLSNDTSGCSWLFQPR (CD8a variant 84) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL P50D SDFRRENEGYYFC S ALSNSIMYF SHF VP VFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 28 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 86) KCQVLLSNETSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE P50E GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 29 SQFRVSPLDRTWNLGETVELKCQVLLSNETSGCSWLFQPRG (CD 8 a variant 86) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS P50E DFRRENEGYYFC S ALSNSIMYF SHF VPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV-74- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443SEQ ID NO: 30 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 90) KCQVLLSNITSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE P50I GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 31 SQFRVSPLDRTWNLGETVELKCQVLLSNITSGCSWLFQPRG (CD8a variant 90) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS P50I DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 32 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 98) KCQVLLSNWTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA P50W EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 33 SQFRVSPLDRTWNLGETVELKCQVLLSNWTSGCSWLFQPR (CD8a variant 98) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL P50W SDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGD KPSLSARYV SEQ ID NO: 34 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD 8 a variant 116) KCQVLLSNPSSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE T51S GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 35 SQFRVSPLDRTWNLGETVELKCQVLLSNPSSGCSWLFQPRG (CD 8 a variant 116) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS T51S DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA-75- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 36 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 123) KCQVLLSNPTNGCSWLFQPRGAAASPTFLLYLSQNKPKAA S52N EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 37 SQFRVSPLDRTWNLGETVELKCQVLLSNPTNGCSWLFQPR (CD8a variant 123) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL S52N SDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGD KPSLSARYV SEQ ID NO: 38 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 125) KCQVLLSNPTCGCSWLFQPRGAAASPTFLLYLSQNKPKAA S52C EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 39 SQFRVSPLDRTWNLGETVELKCQVLLSNPTCGCSWLFQPR (CD8a variant 125) GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL S52C SDFRRENEGYYFC S ALSNSIMYF SHF VP VFLP AKPTTTP APR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 40 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 128) KCQVLLSNPTGGCSWLFQPRGAAASPTFLLYLSQNKPKAA S52G EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 41 SQFRVSPLDRTWNLGETVELKCQVLLSNPTGGCSWLFQPR (CD8a variant 128) GAAASPTFLLYL SQNKPKAAEGLDTQRF SGKRLGDTFVLTL-76- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443S52G SDFRRENEG YYFC S ALSNSIMYF SHF VP VFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 42 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 137) KCQVLLSNPTTGCSWLFQPRGAAASPTFLLYLSQNKPKAAE S52T GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 43 SQFRVSPLDRTWNLGETVELKCQVLLSNPTTGCSWLFQPRG (CD8a variant 137) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS S52T DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKP SLSARYV SEQ ID NO: 44 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 201) KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE N120A GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSASIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 45 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG (CD8a variant 201) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS N120A DFRRENEGYYFCSALSASIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 46 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 218) KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE N120W GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSWSIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV-77- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443SEQ ID NO: 47 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG (CD8a variant 218) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS N120W DFRRENEGYYFCSALSWSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 48 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 227) KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE S121Q GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNQIM YF SHF VPVFLPAKPTTTPAPRPPTPAPTIASQPL SLRPE ACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 49 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG (CD8a variant 227) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS S121Q DFRRENEGYYFCSALSNQIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 50 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 230) KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE S121I GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNIIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 51 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG (CD8a variant 230) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS S121I DFRRENEGYYFCSALSNIIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 52 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL (CD8a variant 239) KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE S121Y GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNYIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP-78- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 53 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG (CD8a variant 239) AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS S121Y DFRRENEGYYFCSALSNYIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 57 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL S121N KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAE GLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNNIM YFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN HRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 58 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG S121N AAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLS DFRRENEGYYFCSALSNNIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYV SEQ ID NO: 59 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL S48A KCQVLLANPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAA EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPVVKSGDKPSLSARYV SEQ ID NO: 60 SQFRVSPLDRTWNLGETVELKCQVLLANPTSGCSWLFQPR S48A GAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTL SDFRRENEG YYFC S ALSNSIMYF SHF VP VFLP AKPTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGD KPSLSARYV SEQ ID NO: 61 MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVEL N76M KCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQMKPKAA-79- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443EGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSI MYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC NHRNRRRVCKCPRPWKSGDKPSLSARYV SEQ ID NO: 62 SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRG N76M AAASPTFLLYLSQMKPKAAEGLDTQRFSGKRLGDTFVLTLS DFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRP PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWA PLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKP SLSARYVEQUIVALENTS

[0237] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0238] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0239] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which -80- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.[0240| All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.4915-4673-0127.1

Claims

Atty. Dkt. No.: 115872-3443CLAIMS1. An engineered cytotoxic T cell thata. comprises a T cell receptor (TCR) that binds to a target antigen and / or a nucleic acid encoding the T cell receptor;b. lacks detectable expression or activity of a wild-type CD8 alpha polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3; and c. comprises a non-endogenous expression vector that includes a nucleic acid sequence encoding a mutant CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62, optionally wherein the nucleic acid sequence of the mutant CD8 alpha polypeptide is operably linked to an expression control sequence.

2. The engineered cytotoxic T cell of claim 1, wherein the TCR is a native TCR, a non-native TCR, or a mimic TCR.

3. The engineered cytotoxic T cell of claim 1 or 2, wherein the TCR is IG4, DMF5, TCR4, MAGE A3, or A6.

4. The engineered cytotoxic T cell of any one of claims 1-3, wherein the mutant CD8 alpha polypeptide exhibits reduced binding to major histocompatibility complex (MHC) relative to the wild-type CD8 alpha polypeptide.

5. The engineered cytotoxic T cell of any one of claims 1-4, wherein the engineered cytotoxic T cell comprises a deletion, an inversion, a missense mutation, a nonsense mutation, or a frameshift mutation in a nucleic acid sequence encoding the wild-type CD8 alpha polypeptide, optionally wherein the nucleic acid sequence encoding the wild-type CD8 alpha polypeptide is SEQ ID NO: 1.

6. The engineered cytotoxic T cell of any one of claims 1-4, wherein the engineered cytotoxic T cell comprises an inhibitory nucleic acid that specifically targets and inhibits the expression of a nucleic acid sequence encoding the wild-type CD8 alpha polypeptide, optionally wherein the nucleic acid sequence encoding the wild-type CD8 alpha polypeptide is SEQ ID NO: 1.

7. The engineered cytotoxic T cell of claim 6, wherein the inhibitory nucleic acid is an antisense oligonucleotide, a siRNA, a sgRNA or a shRNA.-82- 4915-4673-0127.1Atty. Dkt. No.: 115872-34438. The engineered cytotoxic T cell of any one of claims 1-7, wherein the engineered cytotoxic T cell is derived from an autologous donor or an allogeneic donor.

9. An engineered CD4+ helper T cell thata. comprises a T cell receptor that binds to a target antigen and / or a nucleic acid encoding the T cell receptor; andb. comprises a non-endogenous expression vector that includes a nucleic acid sequence encoding a CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62, and is optionally operably linked to an expression control sequence.

10. The engineered CD4+ helper T cell of claim 9, wherein the TCR is a native TCR, a non-native TCR, or a mimic TCR.

11. The engineered CD4+ helper T cell of claim 9 or 10, wherein the TCR is IG4, DMF5, TCR4, MAGE A3, or A6.

12. The engineered CD4+ helper T cell of any one of claims 9-11, wherein the engineered cytotoxic T cell is derived from an autologous donor or an allogeneic donor.

13. The engineered cytotoxic T cell of any one of claims 1-8 or the engineered CD4+ helper T cell of any one of claims 9-12, wherein the non-endogenous expression vector is a plasmid, a cosmid, a bacmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, or a retroviral vector.

14. The engineered cytotoxic T cell of any one of claims 1-8 or 13 or the engineered CD4+ helper T cell of any one of claims 9-13, wherein the expression control sequence is an inducible promoter, a constitutive promoter, a native promoter, or a heterologous promoter.

15. The engineered cytotoxic T cell of any one of claims 1-8 or 13-14 or the engineered CD4+ helper T cell of any one of claims 9-14, wherein the target antigen comprises a tumor antigen.

16. A composition comprising an effective amount of the engineered cytotoxic T cell of any one of claims 1-8 or 13-15 or the engineered CD4+ helper T cell of any one of claims 9-15, and a pharmaceutically acceptable carrier.-83- 4915-4673-0127.1Atty. Dkt. No.: 115872-344317. A kit comprising an expression vector that includes a nucleic acid sequence encoding a CD8 alpha amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62, and instructions for transducing CD4+ helper T cells with the expression vector.

18. A kit comprising an expression vector that includes a nucleic acid sequence encoding a mutant CD8 alpha amino acid sequence of any one of SEQ ID NOs: 4- 53 or 57-62, and instructions for transducing cytotoxic T cells with the expression vector.

19. The kit of claim 17 or 18, further comprising a vector encoding an engineered T- cell receptor (TCR) that binds to a target antigen.

20. A method for treating cancer or inhibiting tumor growth in a subject in need thereof comprising administering to the subject an effective amount of the engineered cytotoxic T cell of any one of claims 1-8 or 13-15, the engineered CD4+ helper T cell of any one of claims 9-15, or the composition of claim 16.

21. A method for mitigating off-target reactivity / toxicity in a subject receiving adoptive T cell therapy comprising administering to the subject an effective amount of the engineered cytotoxic T cell of any one of claims 1-8 or 13-15, the engineered CD4+ helper T cell of any one of claims 9-15, or the composition of claim 16.

22. The method of claim 21, wherein the subject suffers from or is diagnosed with cancer.

23. The method of claim 20 or 22, wherein the cancer or tumor is selected from the group consisting of adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, acute and chronic leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal-84- 4915-4673-0127.1Atty. Dkt. No.: 115872-3443cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.

24. The method of any one of claims 20-23, wherein the engineered cytotoxic T cell or engineered CD4+ helper T cell is administered pleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.

25. A method of preparing cytotoxic T cells for adoptive cell therapy comprising:isolating cytotoxic T cells from a donor subject;inactivating expression and / or activity of a wild-type CD8 alpha polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 in the cytotoxic T cells;transducing the cytotoxic T cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a mutant CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62; andadministering the transduced cytotoxic T cells to a recipient subject.

26. A method of preparing CD4+ helper T cells for adoptive cell therapy comprising:isolating CD4+ helper T cells from a donor subject;transducing the CD4+ helper T cells with a non-endogenous expression vector that includes a nucleic acid sequence encoding a CD8 alpha polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 4-53 or 57-62; andadministering the transduced CD4+ helper T cells to a recipient subject.

27. The method of claim 25 or 26, wherein the donor subject and the recipient subject are the same or different.

28. The method of any one of claims 25-27, wherein the T cells comprise a native T cell receptor (TCR), a non-native TCR, or a mimic TCR.-85- 4915-4673-0127.1

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