Immunotherapy targeting modified KRAS g12v antigen

Abstract

The present disclosure provides compositions and methods for targeting a Ras antigen to, for example, treat or prevent cancer. Disclosed embodiments include binding proteins, such as T cell receptors bind to a Ras antigen:HLA complex, optionally wherein the Ras antigen is the peptide KLVVVGAVGV and comprises zero, one, two, or three methyl groups at a N-terminal lysine thereof, and wherein the HLA is HLA-A*02:01. In some embodiments, the N-terminal lysine comprises one, two, or three methyl groups. In some embodiments, the N-terminal lysine comprises two or three methyl groups. In some embodiments, the N-terminal lysine comprises two methyl groups. In some embodiments, the N-terminal lysine comprises three methyl groups. Disclosed binding proteins are highly sensitive to antigen, capable of inducing activation of host T cells at low concentrations of peptide antigen. In certain embodiments, binding proteins of the present disclosure are non-alloreactive against, are substantially non-alloreactive against, and / or have a low risk of alloreactivity against (i) amino acid sequences from the human proteome. Polynucleotides encoding such binding protein can introduced into a host cell, such as a T cell, and the cell can be used in immunotherapy for treating various cancers.

Description

[0001] IMMUNOTHERAPY TARGETING MODIFIED KRAS G12V ANTIGEN

[0002] BACKGROUND

[0003] Ras family proteins are small GTPases that are involved with transmitting signals within cells, including, for example, transduction of cell proliferation. Exemplary RAS proteins include KRAS (also called C-K-RAS, CFC2, K-RAS2A, K-RAS2B, K-RAS4A, K-RAS4B, KI-RAS, KRAS1, KRAS2, NS, NS3, RALD, RASK2, K-ras, KRAS proto-oncogene, GTPase, and c-Ki- ras2), HRAS, and NRAS. Mutations in RAS proteins that disrupt negative growth signaling can lead to continuous proliferation of the cell. KRAS is one of the most frequently mutated protooncogenes in a variety of human cancers, including melanomas, endometrial, thyroid, pancreatic, colorectal, breast, ovarian, and lung cancers, as well as some instances of myeloid leukemias such as AML. Pharmacological inhibitors have been developed that target KRAS G12C, but primary and adaptive resistance of cancers to these inhibitors has been reported (e.g., Awad et al. NEJM 384:2382-2393 (2021)).

[0004] TCRs targeting KRAS epitopes containing a mutation at G12 presented by several HLA alleles including HLA-A3, HLA-A11, and HLA-C8 have been reported, and in vitro tumor recognition and in vivo efficacy have been reported. The alleles for these HLA proteins, however, have low frequency in many human populations.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The contents of the electronic sequence listing (360056-515WO-SL. xml; Size: 142,319 bytes; and Date of Creation: September 9, 2025) is herein incorporated by reference in its entirety.

[0007] BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A shows TCR functional avidity (reported as logl0(EC50) values) of a set of MHC class I-restricted TCRs (PCT Publication No. WO 2020 / 172332, Figure 18C) against a KRAS G12V 5-14 peptide in complex with (z.e., presented by) HLA-A2 (also shown herein as HLA-A*02:01, HLA-A02:01, and A2). Functional avidity was tested by peptide dose-response after a 4hr stimulation with individual peptides and labelling with IFN-y antibody. Lower values indicate higher functional avidity. Figure IB shows functional avidities measured for CD8+ T cells tranduced to express “TCR2” or “TCR19” of Figure 1 A. Figure 1C shows computational prediction, by NetMHCPan 4.1, of ten HLA-A*02:01 -presented KRAS epitopes. Three of the predicted epitopes contain the G12V mutation. As taught herein, two of the predicted epitopes containing the G12V mutation contain a lysine residue. Figure ID summarizes results from immunoprecipitation of whole-protein KRAS from DAN-G and CFPAC1 cells, followed by liquid chromatography-mass spectrometry, showing that in DAN-G cells, but not in CFPAC1 cells, lysine side chain trimethylation was present at lysine-16 - representing full replacement of all hydrogens of the amine group by methyl groups. Lysine-5 was not recoverable or detectable in this data set, likely due to the cleavage of KRAS protein by trypsin immediately after lysine-5 during sample processing for mass spectrometry.

[0009] Figures 2A-2B show the ability of CD8+ T cells expressing the indicated TCR (TCR delivered to T cells by lentivirus as a codon-optimized P2A-linked epression construct) to kill live KRAS G12V- and HLA-A2-expressing CFPAC1 (Figure 2A) and DAN-G (Figure 2B) pancreatic adenocarcinoma cells when cocultured at a 4: 1 T celktumor cell ratio. Cocultures were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; “NR” (Figure 2A) indicates red fluorescent protein; “NG” (Figure 2B) indicates green fluorescent protein.

[0010] Figure 3 is a heatmap comparing functional avidity (reported as -logl0(EC50) IFN-y values) of CD8+ T cells expressing TCR2 or TCR19 to bind the indicated synthetic KRAS G12V-containing peptides in complex with HLA-A2. Tested peptides included KRAS G12V 5- 14 with and without mono-methylation of the lysine-5 side chain. Higher values indicate higher functional avidity. EC50 values were calculated from T cell exposure to peptide concentrations ranging from 1 pg / ml to 10'5pg / ml. Except for the bottom-right square in the heatmap and the four squares at the top of the heatmap, the squares indicate EC50 calculations that lacked a stable fit, e.g., due to lack of response to peptide even at high doses.

[0011] Figure 4 shows (top) certain predicted KRAS peptide epitopes determined using NetMHCPan 4.0 and (bottom) ARTEMIS mass spectrometry data of peptides eluted from HLA- A2 from cell lines. ARTEMIS data is for peptides eluted from HLA-A2. Data is from the 293F cell line, as commonly used for proteomics assays requiring high protein expression. All cell lines were transduced with an HLA-A2 single chain secreted dimer as well as a KRAS G12V constitutive expression construct to increase presentation of a KRAS G12V peptide:HLA complex, and the likelihood of detecting a presented peptide antigen by mass spectrometry. The first 100 amino acids of KRAS G12V are shown. Epitopes predicted by NetMHCPan 4.0 are as indicated; epitopes detected in cell lines by ARTEMIS are as indicated. Figure 5 presents data relating to reconstitution of properly folded surface HLA-A2 expression on T2 cells after exogenous loading with the indicated wild type KRAS peptide, KRAS G12D peptide, or KRAS G12V peptide without lysine 5 side chain methylation, or with mono- (“...meK”), di- . ,me(2K)”), or tri -methylated . ,me(3)K”) lysine 5, at the indicated concentrations. Only the right-most setting (“no peptide”) received 0 micrograms / mL peptide.

[0012] Figures 6A-6B show structural modeling, generated using Rosetta, of KRAS G12V 5- 14:HLA-A*02:01 complexes for unmethylated KRAS G12V peptide, and KRAS G12V peptide in which lysine 5 is mono-, di-, or tri-methylated, as indicated. The lysine-5 side chain is circled in all images. The dot with a black border and a checker pattern inside (approximately within or adjacent to the circle in each panel) serves as a common reference point. (6B) Same models as (6A) but displayed from a perspective showing protrusion of the methylated lysine more clearly.

[0013] Figure 7 presents a heatmap of results from an alanine scan of the KRAS G12V peptide using CD8+ T cells expressing TCR2 or TCR19. Peptides with the indicated individual position substituted with alanine (or, for residue 11 which is already alanine, substituted with either threonine or glycine) were used to pulse (0.1 pg / ml) antigen-presenting cells to stimulate primary human CD8+ T cells expressing the TCR, and subsequent ZFNy expression was measured. Higher IFNy induction indicates greater tolerance for amino acid substitution at that position. X-axis numbers indicate the amino acid number in the KRAS G12V protein, and letters indicate the amino acid substitution. A: alanine; G: glycine; T: threonine. Last column is unmodified KRAS G12V 5-14.

[0014] Figure 8 is a diagram of the workflow used for structural prediction of TCR:(peptide:HLA complex) complexes using a combined Alphafold-Rosetta pipeline. Alphafold allows for deep learning-based prediction of proteins and protein complexes, which was applied to predict TCR:(peptide:HLA complex) complex structure. However, the Alphafold structure did contain methylation of the peptide, which Alphafold cannot incorporate reliably. The Alphafold structure was used as input into Rosetta, which used physics-based energy functions to model methylation and reoptimize the structure.

[0015] Figure 9 is a structural model, as predicted by a combined Alphafold-Rosetta pipeline of Figure 8, of a ‘core’ CDR3a sequence of an existing TCR that specifically binds to KRAS G12V 5-14 (TCR2) and its relation to the lysine-5 side chain of KRAS G12V 5-14 in complex with HL A-A* 02:01.

[0016] Figure 10 shows tetramer binding patterns of KRAS G12V 5-14:HLA-A2 complexes with unmethylated or methylated lysine 5 side chains for three different mutagenesis libraries based on TCR2. Mutagenesis libraries were generated by mutagenesis of (only) the core CDR3a sequence computationally identified as shown in Figure 9, followed by transduction of the TCRs into Nur77-GFP reporter Jurkat cells and subsequent clonal expansions. Tetramer binding patterns were measured after clonal expansion.

[0017] Figures 11A-11B relate to certain TCRs with modified CDR3a sequences identified from the libraries assessed as described for Figure 10. Figure 11A provides mutated variant sequences of the TCR2 CDR3a (IMGT-JUNCTION definition) identified from isolated sorted clones of the mutagenesis library that exhibit the peptide recognition noted in the figure. Figure 11B is a heatmap showing -logl0(EC50) values calculated from dose response assays using the indicated TCRs derived from the mutagenesis library assessed in Figure 10, or the original TCR2. TCR binding to KRAS G12V 5-14:HLA-A2 complexes with unmethylated lysine 5, or mono-, di-, or tri- methulated lysine 5 was assessed. The light gray squares in the bottom-right -third of the heatmap (e.g., TCR 4UM against wild-type or G12D; TCR EDVA against monomethylated, demethylated, trimethylated, G12D, or wild-type) indicate EC50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses. TCR 4UM (TCR EDRT, meaning a change from the TCR2 CDR3a “EDNT” core sequence to “EDRT”) is the 4th TCR clonotype identified from an initial sequencing dataset from sorted Jurkat cells binding to both unmethylated (“U”) and methylated (“M”) tetramers. Other TCRs are labeled by changes in the core TCR2 CDR3a amino acid sequence (e.g., from EDNT to EDST).

[0018] Figures 12A-12B relate to dose response curves of CD8+ T cells expressing the identified TCR after exposure to wild type KRAS 5-14 (Figure 12A) or KRAS G12D 5-14 (Figure 12B) peptides presented by HLA-A2.

[0019] Figure 13 relates to structural predictions for a TCR2:(KRAS G12V 5-14: HLA-A2 complex) complex for unmethylated and mono-, di-, and tri- methylated KRAS G12V 5-14, as well as unmethylated KRAS wild type or KRAS G12D. The CDR3a core sequence is shown as individual amino acids within the TCR.

[0020] Figure 14 relates to the same type of structural predictions as in Figure 13, but for TCR EDST. Complexes are illustrated for unmethylated and mono-, di-, and tri- methylated variants of KRAS G12V 5-14, as well as unmethylated KRAS wild type or KRAS G12D. The CDR3a core sequence is shown as individual amino acids within the TCR. The images from the same KRAS variants from Figure 13 are overlaid with transparency to facilitate visual comparison. With TCR EDST, residues in the core sequence of CDR3a and the lysine-5 side chain of each variant of KRAS G12V peptide appear closer to each other than with TCR2. Figures 15A and 15B provide data showing the ability of CD8+ T cells expressing TCR2, TCR EDST, or TCR EDRS to kill DAN-G (Figure 15A) or CFPAC1 (Figure 15B) pancreatic adenocarcinoma cells expressing HLA-2 and KRAS G12V 5-14 in cocultures. The CD8+ T cells expressing these TCRs were also cocultured with the HeLa cervical carcinoma cell line, which expresses wild type KRAS (Figure 15C) or the Panel pancreatic adenocarcinoma, which expresses KRAS G12D (Figure 15D). Cocultures of T cells and tumor cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR (Figure 15B and Figure 15D) is red fluorescent protein; NG (Figure 15A and Figure 15C) is green fluorescent protein.

[0021] Figure 16 relates to results of an alanine scan of KRAS G12V 5-14 peptide tested against TCR2 and the indicated engineered variants thereof. Each panel indicates the resulting expression of Nur77-GFP upon replacement of a KRAS G12V peptide residue with alanine (or in the case of alanine-11, replacement with either glycine or threonine). Alanine scans were performed using 0.1 pg / ml peptide concentrations, with tolerance to a change defined as retaining >35% of responding cells positive for Nur77-GFP reporter expression, there were distinct positions of the epitope that tolerated amino acid replacements and remained stimulatory.

[0022] Figure 17 shows fFNy secretion by CD8+ T cells expressing TCR2 or the indicated engineered TCR2 variant, after exposure to 0.1 pg / ml of potential off-target synthetic peptides from the human proteome fitting the K-x-x-V-V-x-A-x-x-x tolerance pattern identified from the alanine scan of Figure 16. Results are from concentration of 0.1 pg / ml for each peptide.

[0023] Figures 18A-18D relate to dose response curves of CD8+ T cells expressing TCR2 or the indicated TCR2 variant TCR after exposure to the potential human off-target peptides tested in Figure 17 to generate the three largest responses at 0.1 pg / ml: CFA6123-32 / 607-616 / 667-676 (Figure 18B), RSLBB35-44 (Figure 18C), and TRXR1342-351 (Figure 18D), compared to the KRASG12V 5-14 peptide (Figure 18A).

[0024] Figure 19 relates to tetramer binding patterns of primary human T cells stimulated with unmethylated and mono- or tri-methylated KRAS G12V peptide antigen presented by HLA-A2. Plots also indicate gates used to sort cells with different avidities for unmethylated and mono- or tri-methylated KRAS G12V 5-14 peptide.

[0025] Figure 20 shows a dose response assay heatmap of-logl0(EC50) fFNy for the indicated TCRs against unmethylated and methylated KRAS G12V, or wild type KRAS, or KRAS G12D peptide presented by HLA-A2. Higher values indicate higher functional avidity. Gray squares corresponding to G12D mutation, and to wild-type for TCRs A2UoMl-l, A2UoMl-2, and A2UoMl-4, indicate EC50 values that lacked a stable fit, e.g., due to limited response to peptide even at high doses.

[0026] Figures 21A and 21B relate to the ability of CD8+ T cells expressing the indicated TCR to kill DAN-G (Figure 21 A) or CFPAC1 (Figure 21B) pancreatic adenocarcinoma cells expressing HLA-2 and KRAS G12V 5-14 in cocultures. Cocultures of fluorescently labeled tumor cells and TCR-T cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR (Figure 21B) is red fluorescent protein; NG (Figure 21A) is green fluorescent protein.

[0027] Figure 22A shows an experimental workflow to enrich and characterize T cell clones with TCRs recognizing a defined peptide epitope.

[0028] Figures 22B and 22C show dose response curves for TCRs against wild type KRAS (Figure 22B) and KRAS G12D 5-14 (Figure 22C) peptides presented by HLA-A2.

[0029] Figures 23A and 23B relate to the ability of CD8+ T cells expressing the indicated TCR to kill HeLa cells expressing wild type KRAS (Figure 23A) or Panc-1 cells expressing KRAS G12D (Figure 23B) in cocultures. Cocultures of fluorescently labeled tumor cells and TCR-T cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NR (Figure 23B) is red fluorescent protein; NG (Figure 23A) is green fluorescent protein.

[0030] Figure 24 relates to results of an alanine scan of KRAS G12V 5-14 peptide tested against T cells expressing TCR A2UoMl-l. Each panel indicates the resulting expression of Nur77- GFP upon replacement of a KRAS G12V residue with alanine (or in the case of alanine- 11, replacement with either glycine or threonine).

[0031] Figure 25 relates to dose response curves of CD8+ Nur77-GFP Jurkat cells expressing TCR A2UoMl-l after exposure to EPIPL 1948- 1957, the potential human off-target peptide as determined from alanine scan results of Figure 24.

[0032] Figures 26A-26D relate to Alphafold-Rosetta structural predictions for TCR A2UoMl- 1 :(KRAS G12V 5-14:HLA-A*02:01 complex) complexes in which KRAS G12V is unmethylated (Figure 26A), mono-(Figure 26B), di-(Figure 26C), or tri-(Figure 26D methylated. TCR A2UoMl-l was isolated from expansion of responding primary human CD8+ T cells from normal repertoires. The four TCR amino acids in closest proximity to the lysine-5 side chain of KRAS G12V are shown with individual molecular bonds.

[0033] Figure 27A: Worfklow for a gene knockout screen for methylation-related genes. DAN- G pancreatic adenocarcinoma cells were transduced with a CRISPR knockout library targeting methylation-related genes, and then cocultured with CD8+ T cells expressing either TCR2 or mutagenized TCR EDST, which respectively have weak or strong recognition of methylated KRAS G12V epitopes. Tumor cells surviving at the end of coculture were sequenced to identify gRNA enrichment or depletion associated with immune evasion. Figure 27B: volcano plot summarizing log2(fold change) of gRNA read frequencies targeting individual genes recovered after coculture of DAN-G cells with T cells expressing either TCR2 or TCR EDST. The threshold of significance was set at loglO(p) < 1.2 (p < 0.05) and is denoted by the horizontal dashed line. Mean counts of all gRNAs per gene were used to calculate fold change values. The data point for gRNAs targeting SUPT6H is specifically labeled at far left. The plot shows knockouts depleted or enriched after cocultures using TCR EDST (upper left quadrant) or TCR2 (upper right quadrant).

[0034] Figures 28A and 28B relate to the ability of CD8+ T cells expressing the indicated TCR to kill DAN-G without SUPT6H knockout (original DAN-G cells) (Figure 28A) or with SUPT6H knockout (Figure 28B). Cocultures of fluorescently labeled tumor cells and TCR-T cells were recorded by Incucyte imaging. Tumor cells expressed nuclear fluorescent protein; NG is green fluorescent protein.

[0035] Figures 28C and 28D: Kaplan-Meier survival plots for individuals who have tumors with KRAS mutations, using public TCGA data. (28C) only tumors with the KRASG12V mutant, with or without concurrent SUPT6H mutation. (28D) any mutant KRAS, with or without a concurrent SUPT6H mutation in the tumor.

[0036] Figure 29: Location of the gRNA binding region in the SUPT6H gene.

[0037] Figure 30: Summary of the genomic position of the commonly occurring mutations detected from DAN-G cells transduced with lentiCRISPRv2 with gRNA targeting SUPT6H. Dots indicate a mutation detected from long-read sequencing.

[0038] Figure 31 shows surface expression of HLA-A2 in DAN-G cells with and without SUPT6H knockout.

[0039] Figure 32 is a Western blot of total expressed KRAS protein in DAN-G cells with and without SUPT6H knockout. KRAS expression in other pancreatic adenocarcinoma cell lines Panc-1 and CFPAC1 is shown for additional comparison. GAPDH protein expression was used as a control.

[0040] DETAILED DESCRIPTION

[0041] The present disclosure generally relates to binding proteins specific for Ras neoantigens, modified host (e.g., immune) cells expressing the same, polynucleotides that encode the binding proteins, and related uses. Mutated Ras proteins (e.g., KRAS, NRAS, HRAS) can produce neoantigens, including a G~>V mutation at position 12 of the full-length KRAS protein (SEQ ID NO: 1; UniProt KB P01116) or at position 12 of the full-length NRAS protein (SEQ ID NO: 3; Uniprot KB P01111) or at position 12 of the full-length HRAS protein (SEQ ID NO:4; Uniprot KB P01112).

[0042] The present disclosure identifies a RAS protein (e.g., KRAS) G12V 5-14 peptide (SEQ ID NO: 2) as able to form a peptide antigen:HLA complex with the prevalent HLA-A*02:01 allele, shows that at least some cancer cells present a post-translationally modified RAS e.g., KRAS) G12V 5-14 peptide (KLVVVGAVGV; comprising one, two, or three methyl groups added to the lysine (K)-5 residue side chain) in the context of HLA-A* 02:01, and provides binding proteins e.g., TCRs having engineering in a CDR or TCRs having CDRs from the natural human repertoire) capable of binding a RAS e.g., KRAS) G12V 5-14 peptide (KLVVVGAVGV): HLA-A*02:01 complex in which the N-terminal lysine of the peptide is, for example: unmethylated, unmethylated or monomethylated; monomethylated or unmethylated; dimethylated; monomethylated, dimethylated, or trimethylated; unmethylated; monomethylated, dimethylated, or trimethylated; or monomethylated or dimethylated. In other words, certain binding proteins can bind a KRAS G12V 5-14 peptide (KLVVVGAVGV): HLA-A*02:01 complex wherein the N-terminal lysine residue of the peptide is in an unmethylated state and / or is in a methylated state.

[0043] In certain embodiments, a binding protein is capable of binding to a RAS (e.g., KRAS) G12V 5-14 peptide (KLVVVGAVGV): HLA-A2 complex in which the N-terminal lysine of the peptide bears: 0 methyl groups; 0 or 1 methyl groups; 1 methyl group; 1 or 2 methyl groups; or 1, 2, or 3 methyl groups.

[0044] Disclosed binding proteins are highly sensitive to antigen, being capable in certain embodiments of inducing activation of host T cells at low concentrations of peptide antigen.

[0045] In certain embodiments, a binding protein comprises a T cell receptor (TCR), such as a membrane-anchored or soluble TCR, a single-chain T cell receptor variable domain fragment (scTv), a single-chain TCR (scTCR), a TCR-CAR, a disulfide-stabilized T cell receptor variable domain fragment (dsTv), a TCR-mimic antibody, or a fusion protein comprising any one or more of the foregoing.

[0046] Disclosed embodiments include binding proteins that are highly selective and display favorable safety properties. In certain embodiments, binding proteins of the present disclosure are non-alloreactive against, are substantially non-alloreactive against, and / or have a low risk of alloreactivity against other peptides from the human proteome.

[0047] In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) is capable of killing CFPAC-1 cells in vitro. In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) is capable of killing DANG cells in vitro.

[0048] In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) does not produce, or does not substantially produce, IFN-y whein in the presence of HLA-A* 02:01 -positive antigen-presenting cells presenting a KRAS wild-type 5-14 peptide in vitro. In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) does not produce, or does not substantially produce, IFN-y whein in the presence of HLA-A*02:01 -positive antigen-presenting cells presenting a KRAS G12D 5-14 peptide in vitro.

[0049] In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) does not kill, or does not substantially kill, HeLa cells expressing wild-type KRAS in vitro. In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) does not kill, or does not substantially kill, Panc-1 cells expressing KRAS G12D in vitro.

[0050] In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) produces no IFN-y, or produces less IFN-y (than when in the presence of HLA-A* 02:01 -positive antigen-presenting cells presenting a KRAS G12V 5-14 peptide) when in the presence of HLA-A* 02:01 -positive antigen-presenting cells presenting a peptide as shown in Figure 17.

[0051] In some embodiments, a T cell (e.g., a human CD8+ or CD4+ T cell) expressing or encoding a binding protein of the present disclosure (e.g., a TCR) is not activated (e.g., as assessed by Nur77 expression, or by expression of GFP under the control of a Nur77 promoter, as described for Figure 25) in the presence of HL A- A* 02:01 -positive antigen-presenting cells presenting an EPIPL 1948-1957 peptide.

[0052] In certain embodiments, host cells expressing a binding protein may exhibit different activation and killing of tumor or cancer cells depending on whether a KRAS G12V peptide is methylated or unmethylated, or both, and, in some emboduments, whether methylation is mono-, di-, or tri- methylation, particularly on the side chain of lysine-5.

[0053] Also provided are polynucleotides and vectors that encode a binding protein, as well as host cells (e.g., immune cells, such as T cells) that encode or express a binding protein, and compositions that comprise a binding protein, host cell, polynucleotide, and / or vector. Also provided are uses of a disclosed binding protein, polynucleotide, vector, host cell, and / or composition to treat a disease or disorder (e.g., a cancer) in a subject, wherein the disease or disorder is characterized by cells that encode a KRAS G12V mutation and express HLA- A*02:01. In some embodiments, a method of treating the disease or disorder comprises administering to a subject a disclosed binding protein, polynucleotide, vector, host cell, and / or composition and further administering to the subject an agent that inhibits expression or activity of SUPT6H.

[0054] Also provided are uses of a binding protein that recognizes a methylated (mono-, di-, and / or tri-) RAS (e.g., KRAS) G12V peptide for determining whether a sample comprises the methylated peptide. The methylated peptide can be provided in a complex with an HLA- A*02:01, and binding can be determined by any appropriate assay, such as an assay described herein, and / or can be indirectly determined by assaying activity (e.g., activation) of a host cell (e.g., a T cell) expressing the binding protein (e.g., a TCR, a TCR-CAR, a scTCR).

[0055] Also provided are methods of screening a binding protein (or a set or library of binding proteins) to determine whether the binding protein binds a Ras (e.g., KRAS) G12V 5-14 peptide comprising one or more (e.g., one, two, three, or more) methyl groups at the N-terminal lysine of the peptide. In some embodiments, the method comprises instroducing the binding protein, or a cell (e.g., a human T cell) expressing the binding protein, to the peptide (e.g., the peptide in complex with an HLA-A*02:01, optionally to a cell presenting the peptide in complex with the HLA-A*02:01). Binding can be determined by any appropriate assay, such as an assay described herein, and / or can be indirectly determined by assaying activity (e.g, activation) of a host cell (e.g, a T cell) expressing the binding protein (e.g., a TCR, a TCR-CAR, a scTCR).

[0056] Also provided are methods of identifying a binding protein that binds a Ras (e.g., KRAS) G12V 5-14 peptide comprising one or more (e.g., one, two, three, or more) methyl groups at the N-terminal lysine of the peptide. In some embodiments, the method comprises instroducing the binding protein, or a cell (e.g., a human T cell) expressing the binding protein, to the peptide (e.g., the peptide in complex with an HLA-A*02:01, optionally to a cell presenting the peptide in complex with the HLA-A*02:01), and determining whether binding occurs. Binding can be determined by any appropriate assay, such as an assay described herein, and / or can be indirectly determined by assaying activity (e.g., activation) of a host cell (e.g., a T cell) expressing the binding protein (e.g., a TCR, a TCR-CAR, a scTCR).

[0057] Also provided are immunogenic compositions that comprise a Ras (e.g, KRAS) G12V 5- 14 peptide in monomethylated, demethylated, or trimethylated form. The immunogenic compositions can further comprise an adjuvant. The immunogenic compositions may be used to, for example, elicit an immune response in a subject (e.g, a human subject), to screen T cells for T cells expressing TCRs that recognize a methylated Ras (e.g., KRAS) G12V 5-14 peptide, and to activate T cells expressing TCRs that recognize a methylated Ras (e.g., KRAS) G12V 5-14 peptide. Also provided are methods of using a presently disclosed binding protein to determine whether a subject expresses a Ras (e.g., KRAS) G12V peptide (preferably a G12V 5-14) comprising one or more methyl groups at a N-terminal lysine thereof.

[0058] Also provided are methods that comprise introducing a polynucleotide encoding a presently disclosed binding protein (or introducing a vector comprising the polynucleotide) into a host cell or into a plurality or population or sample of host cells. The vector or polynucleotide can, for example, be comprised in a composition (e.g., comprising a lipid nanoparticle). In some embodiments, the polynucleotide or vector further encodes: a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain, or both. In certain embodiments, the host cell or cells comprises a T cell, such as a CD4+ T cell or a CD8+ T cell. In certain embodiments, the host cell or cells comprises a primary T cell. In certain embodiments, the host cell or cells comprises a peripheral blood mononuclear cell (PBMC). In certain embodiments, the host cell or cells comprises a hematopoietic progenitor cell. In certain embodiments, the host cell or cells comprises a stem cell. In certain embodiments, the host cell or cells comprises a pluripotent stem cell, optionally an induced pluripotent stem cell. In some embodiments, a method further comprises culturing the host cell or cells. In some embodiments, the host cell or cells is from a subject having a disease or disorder associated with a KRAS G12V or NRAS G12V or HRAS G12V mutation. In some embodiments, the disease or disorder comprises a cancer. In some embodiments, the subject is positive for expression of an HLA-A2, such as HLA-A*02:01. In certain embodiments, the host cell or cells is from a healthy subject. In some embodiments, the method is performed in vitro. In other embodiments, the method is performed ex vivo. Also provided is a host cell, host cell population, or host cell sample made by the method. In some embodiments, a host cell population comprises CD8+ T cells, CD4+ T cells, or both. In some embodiments, a method further comprises selecting for and combining CD8+ T cells with CD4+ T cells to provide a composition that comprises the CD8+ T cells and CD4+ T cells in about a 1:1 ratio.

[0059] Certain Embodiments provide as follows:

[0060] Embodiment 1. A binding protein that is capable of binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing 2 or 3 methyl groups.

[0061] Embodiment 2. The binding protein of Embodiment 1, wherein the N-terminal lysine of the peptide comprises a side chain bearing 2 methyl groups.

[0062] Embodiment 3. The binding protein of Embodiment 1, wherein the N-terminal lysine of the peptide comprises a side chain bearing 3 methyl groups.

[0063] Embodiment 4. The binding protein of any one of Embodiments 1-3, wherein the binding protein is also capable of binding to peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing 0 or 1 methyl groups.

[0064] Embodiment 5. The binding protein of any one of Embodiments 1-4, wherein the binding protein comprises a binding domain that binds the peptide:HLA complex, wherein the binding domain optionally comprises an immunoglobulin superfamily variable domain.

[0065] Embodiment 6. The binding protein of Embodiment 5, wherein binding domain comprises a TCR a-chain variable domain (Va) and / or a TCR P-chain variable domain (VP), wherein the binding domain optionally comprises the Va domain and the VP domain.

[0066] Embodiment 7. The binding protein of any one of Embodiments 1-9, comprising a heavy chain variable domain (VH) and / or a light chain variable domain (VL) of a TCR-mimic antibody, wherein the binding domain optionally comprises the VH domain and the VL domain.

[0067] Embodiment 8. A binding protein comprising a TCR a-chain variable domain (Va) and a TCR P-chain variable domain (VP), wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV, the N-terminal lysine of the peptide bears zero, one, two, or three methyl groups, and the HLA is HLA-A*02:01, and wherein the Va domain comprises complementarity determining region (CDR)la, CDR2a, and CDR3a amino acid sequences and the VP domain comprises CDRip, CDR2P, and CDR3P amino acid sequences, wherein the CDRla, CDR2a, CDR3a CDRip, CDR2P, and CDR3P amino acid sequences are: (i) DSSSTY, IFSNMDM, CAEIEDRTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively; (ii) DSSSTY, IFSNMDM, CAEIEDSTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively;

[0068] (iii) DSSSTY, IFSNMDM, CAEIEDRSDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively; (iv) DSSSTY, IFSNMDM, CAEITGNTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively; (v) DSSSTY, IFSNMDM, CAEIEDVADKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively; (vi) DSSSTY, IFSNMDM, CAEIEARRDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively; (vii) YGATPY, YFSGDTLV, CAVGADSNYQLIW, SGHNS, FNNNVP, and CASSLVFYNEQFF, respectively; (viii) NSAFQY, TYSSGN, CAMSDDWGKLQF, PRHDT, FYEKMQ, and CASSTIAGGRGNEQFF, respectively; or (ix) DSAIYN, IQSSQRE, CAVPPGDDYKLSF, MDHEN, SYDVKM, and CASSPIQRGDQPQHF, respectively.

[0069] Embodiment 10. A T cell receptor comprising an alpha chain and a beta chain, wherein: The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDRT (4UM), as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDRT (4UM), as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDST, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDST, as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDRS, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDRS, as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR TGNT, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR TGNT, as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDVA, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDVA, as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EARR, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EARR, as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-l, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-l, as shown in Table X; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-2, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-2, as shown in Table X; or The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l- 2, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-2, as shown in Table X.

[0070] Embodiment 11. A T cell receptor comprising an alpha chain and a beta chain, wherein: The alpha chain comprises the alpha chain variable domain TCR EDRT (4UM), as shown in Table Y, and the beta chain variable domain of TCR EDRT (4UM), as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EDST, as shown in Table Y, and the beta chain variable domain of TCR EDST, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EDRS, as shown in Table Y, and the beta chain variable domain of TCR EDRS (4UM), as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR TGNT, as shown in Table Y, and the beta chain variable domain of TCR TGNT, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EDVA, as shown in Table Y, and the beta chain variable domain of TCR EDVA, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EARR, as shown in Table Y, and the beta chain variable domain of TCR EARR, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR A2UoM-l-l, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-l, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR A2UoM-l-2, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-2, as shown in Table Z; or The alpha chain comprises the alpha chain variable domain TCR A2UoM-l-4, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-4, as shown in Table Z.

[0071] Embodiment 12. A binding protein comprising a TCR alpha chain variable domain of any one of the TCRs shown in Table Y and the corresponding beta chain variable domain shown in Table X.

[0072] Embodiment 13. The binding protein or T cell receptor of any one of Embodiments 1-12, further comprising a TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP).

[0073] Embodiment 14. The binding protein or T cell receptor of Embodiment 13, wherein the Ca comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.:18, 19, 44, 45, and 69. Embodiment 15. The binding protein or T cell receptor of Embodiment 13 or 14, wherein the Cp comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in any one of SEQ ID NOs.: 28, 29, 54, 55, and 70-73.

[0074] Embodiment 16. The binding protein or T cell receptor of any one of Embodiments 13-15, wherein the Ca and the CP comprise or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequences set forth in SEQ ID NOs.: (i) 18 and 28, respectively; (ii) 19 and 29, respectively; (iii) 44 and 54, respectively; or (iv) 45 and 55, respectively.

[0075] Embodiment 17. The binding protein or T cell receptor of any one of Embodiments 13-16, wherein the Ca, the CP, or both comprise modification(s) that promote preferential pairing of the Ca to the Cp.

[0076] Embodiment 18. The binding protein or T cell receptor of any one of Embodiments 13-16, wherein the Ca and the CP each comprises an introduced cysteine residue that promotes preferential pairing of the Ca to the Cp.

[0077] Embodiment 19. The binding protein or T cell receptor of any one of Embodiments 13-16, wherein the Ca comprises a T48C substitution and the CP comprises a S57C substitution to promote preferential pairing of the Ca to the Cp.

[0078] Embodiment 20. The binding protein or T cell receptor of any one of Embodiments 1-19, comprising a TCR a chain and a TCR P chain, wherein the TCR a chain and the TCR P chain comprise or consist of amino acid sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequences set forth in: (i) SEQ ID NOs.: 12 and 22, respectively; (ii) SEQ ID NOs.: 20 and 30, respectively; (iii) SEQ ID NOS.: 12 and 30, respectively; (iv) SEQ ID NOs.:20 and 22, respectively; (v) SEQ ID NOs.:38 and 48, respectively; (vi) SEQ ID NOs.: 46 and 56, respectively; (vii) SEQ ID NOs.:38 and 56, respectively; or (viii) SEQ ID NOs.:46 and 48, respectively.

[0079] Embodiment 21. The binding protein or T cell receptor of any one of Embodiments 1-20, wherein the binding protein comprises a TCR, a single-chain TCR (scTCR), a single-chain T cell receptor variable fragment (scTv), or a chimeric antigen receptor (CAR). Embodiment 22. The binding protein of Embodiment 21, wherein the binding protein comprises a TCR.

[0080] Embodiment 23. The binding protein of any one of Embodiments 1-22, wherein the binding protein comprises an EC50 of at most 100 nM, at most 50 nM, at most 25 nM, at most 10 nM, at most 1 nM, at most 750 pM, at most 500 pM, at most 250 pM, at most 100 pM, at most 75 pM, or at most 60 pM in a CD 137 surface expression assay for functional avidity to the peptide.

[0081] Embodiment 24. An isolated polynucleotide encoding the binding protein or T cell receptor of any one of Embodiments 1-23.

[0082] Embodiment 25. [reserved].

[0083] Embodiment 26. The polynucleotide of Embodiment 24 or 25, further comprising:

[0084] (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 coreceptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii)a polynucleotide of (i) and a polynucleotide of (ii).

[0085] Embodiment 27. The polynucleotide of Embodiment 26, comprising:

[0086] (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co- receptor a chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor p chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).

[0087] Embodiment 28. The polynucleotide of Embodiment 26 or 27, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between:

[0088] (1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; and / or

[0089] (2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain.

[0090] Embodiment 29. The polynucleotide of any one of Embodiments 26-28, comprising, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnBP);

[0091] (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCPi)-(pnCD8a)- (pnSCP2)-(pnCD8P); (iv) (pnBP)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnCD8a);

[0092] (v) (pnCD8a)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8P); or (vi) (pnCD8P)-(pnSCPi)- (pnBP)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein pnBP is the polynucleotide encoding a binding protein, and wherein pnSCPi and pnSCP? are each independently a polynucleotide encoding a selfcleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0093] Embodiment 30. The polynucleotide of any one of Embodiments 26-29, wherein the encoded binding protein comprises a TCRa chain and a TCRP chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.

[0094] Embodiment 31. The polynucleotide of Embodiment 30, comprising, operably linked in-frame:

[0095] (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa);

[0096] (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa);

[0097] (iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP);

[0098] (iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP);

[0099] (v) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)-(pnCD8a)-(pnSCP3)-(pnCD8P);

[0100] (vi) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)-(pnCD8P)-(pnSCP3)-(pnCD8a);

[0101] (vii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8a)-(pnSCP3)-(pnCD8P);

[0102] (viii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8P)-(pnSCP3)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnTCRa is the polynucleotide encoding a TCR a chain, wherein pnTCRP is the polynucleotide encoding a TCR P chain, and wherein pnSCPi, pnSCP2, and pnSCPs are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0103] Embodiment 32. The polynucleotide of Embodiment 31, wherein the pnSCPi encodes a T2A peptide, the pnSCP2 encodes a P2A peptide, and the pnSCP3 encodes a P2A peptide.

[0104] Embodiment 37. An expression vector, comprising a polynucleotide as disclosed and cliaimed herein operably linked to an expression control sequence. Embodiment 38. The expression vector of Embodiment 37, wherein the expression control sequence comprises an MSCV promoter.

[0105] Embodiment 39. The expression vector of Embodiment 37 or Embodiment 38, wherein the expression control sequence drives expression of a single mRNA encoding the extracellular portion of the CD8 co-receptor a chain, the extracellular portion of the CD8 coreceptor P chain, the TCR a chain, and the TCR P chain.

[0106] Embodiment 40. The expression vector of any one of Embodiments 37-39, wherein the vector is capable of delivering the polynucleotide to a host cell.

[0107] Embodiment 41. The expression vector of Embodiment 40, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

[0108] Embodiment 42. The expression vector of Embodiment 41, wherein the human immune system cell is a CD4+T cell, a CD8+T cell, a CD4 CD8' double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof.

[0109] Embodiment 43. The expression vector of Embodiment 42, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0110] Embodiment 44. The expression vector of any one of Embodiments 37-43, wherein the vector is a viral vector.

[0111] Embodiment 45. The expression vector of Embodiment 44, wherein the viral vector is a lentiviral vector or a y-retroviral vector.

[0112] Embodiment 46. The expression vector of Embodiment 44, wherein the viral vector is a self-inactivating lentiviral vector.

[0113] Embodiment 47. The expression vector of Embodiment 44 or Embodiment 46, wherein the viral vector is a third generation lentiviral vector.

[0114] Embodiment 48. A host cell modified to comprise the polynucleotide of any one of Embodiments 24-36 and / or the expression vector of any one of Embodiments 37-47 and / or to express the binding protein of any one of Embodiments 1-23.

[0115] Embodiment 49. The host cell of Embodiment 48, wherein the modified cell comprises a hematopoietic progenitor cell and / or a human immune cell.

[0116] Embodiment 50. The host cell of Embodiment 49, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. Embodiment 51. The host cell of Embodiment 50, wherein the immune cell comprises a CD4+T cell, a CD8+T cell, a CD4' CD8' double negative T cell, a y5 T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4+T cell and a CD8+T cell, wherein, further optionally, the CD4+T cell, the CD8+T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

[0117] Embodiment 52. The host cell of any one of Embodiments 48-51, wherein the modified cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.

[0118] Embodiment 53. The host cell of Embodiment 52, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.

[0119] Embodiment 54. The host cell of Embodiment 52 or 53, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.

[0120] Embodiment 55. A composition comprising the host cell of any one of Embodiments 48-54 and a pharmaceutically acceptable carrier, diluent, or excipient.

[0121] Embodiment 56. The composition of Embodiment 55, comprising at least about 30% modified CD4+T cells, combined with (ii) a composition comprising at least about 30% modified CD8+T cells, in about a 1 : 1 ratio.

[0122] Embodiment 57. The composition of Embodiment 55 or 56, wherein the composition contains substantially no naive T cells.

[0123] Embodiment 58. A composition comprising:

[0124] (ix) the binding protein of any one of Embodiments 1-23;

[0125] (x) the polynucleotide of any one of Embodiments 24-36; (xi) the expression vector of any one of Embodiments 37-47; and / or

[0126] (xii) the host cell of any one of Embodiments 48-54, and a pharmaceutically acceptable carrier, excipient, or diluent. Embodiment 59. A method for treating a disease or disorder associated with a KRAS G12V mutation or a NRAS G12V mutation or a HRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of:

[0127] (i) the binding protein of any one of Embodiments 1-23;

[0128] (ii) the polynucleotide of any one of Embodiments 24-36;

[0129] (iii) the expression vector of any one of Embodiments 37-47;

[0130] (iv) the host cell of any one of Embodiments 48-54, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and wherein, optionally, the host cell is autologous, allogeneic, or syngeneic to the subject; and / or

[0131] (v) the composition of any one of Embodiments 55-58.

[0132] Embodiment 60. The method of Embodiment 59, wherein the disease or disorder comprises a cancer, wherein the cancer is optionally a solid cancer or a hematological malignancy.

[0133] Embodiment 61. The method of Embodiment 59 or 60, wherein the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a nonsmall-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

[0134] Embodiment 62. The method of any one of Embodiments 59-61, wherein the binding protein, polynucleotide, vector, host cell, or composition is administered to the subject parenterally or intravenously.

[0135] Embodiment 63. The method of any one of Embodiments 59-62, wherein the method comprises administering a plurality of doses of any one or more of (i)-(v) to the subject.

[0136] Embodiment 64. The method of Embodiment 63, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.

[0137] Embodiment 65. The method of any one of Embodiments 59-64, wherein the composition comprises the host cell or the composition comprising the host cell, and wherein the method comprises administering the host cell or composition to the subject at a dose of about 104cells / kg to about 1011cells / kg.

[0138] Embodiment 66. The method of any one of Embodiments 59-65, wherein the method comprises administering to the subject at least 5xlOA8, at least lxlOA9, at least 5xlOA9, at least lxl0A10, at least 1.5xl0A10, at least 2xl0A10, or at least 5xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

[0139] Embodiment 67. The method of any one of Embodiments 59-65, wherein the method comprises administering to the subject at most 5xlOA9, at most lxl0A10, at most 1.5xl0A10, at most 2x10A10, at most 5xl0A10, at most lxlOAl l, or at most 5xl0Al l viable host cells that comprise the binding protein, optionally in a single dose.

[0140] Embodiment 68. The method of any one of Embodiments 59-65, wherein the method comprises administering to the subject about 5xlOA9, about 6xlOA9, about 7xlOA9, about 8xlOA9, about 9xlOA9, about lxl0A10, about L lxl0A10, about 1.2xl0A10, about 1.3xl0A10, about 1.4xl0A10, about 1.5xl0A10, about 1.6xl0A10, about 1.7xl0A10, about 1.8xl0A10, about 1.9xl0A10, or about 2xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

[0141] Embodiment 69. The method of any one of Embodiments 59-65, wherein the method comprises administering to the subject about 5 xlOA9 to about 1 xlOAl 1, about 5 xlOA9 to about 5 xl0A10, about 5 xlOA9 to about 2 xl0A10, about 5 xlOA9 to about 1.5 xl0A10, about 5 xlOA9 to about 1 xl0A10, about 1 xl0A10 to about 1 xlOAl l, about 1 xl0A10 to about 5 xl0A10, about 1 xl0A10 to about 2 xl0A10, or about 1 xl0A10 to about 1.5 xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

[0142] Embodiment 70. The method of any one of Embodiments 59-69, further comprising determining that the subject expresses HLA-A* 11, optionally HLA-A* 11 :01, prior to administering the binding protein, polynucleotide, vector, host cell, or composition.

[0143] Embodiment 71. The method of any one of Embodiments 59-70, wherein the method further comprises administering a cytokine to the subject.

[0144] Embodiment 72. The method of Embodiment 71, wherein the cytokine comprises IL-2, IL-15, or IL-21.

[0145] Embodiment 73. The method of any one of Embodiments 59-72, wherein the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

[0146] Embodiment 74. The binding protein of any one of Embodiments 1-23, the polynucleotide of any one of Embodiments 24-36, the expression vector of any one of Embodiments 37-47, the host cell of any one of Embodiments 48-54, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and / or the composition of any one of Embodiments 55-58, for use in a method for treating a disease or disorder associated with a KRAS G12V or a NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

[0147] Embodiment 75. The binding protein of any one of Embodiments 1-23, the polynucleotide of any one of Embodiments 24-36, the expression vector of any one of Embodiments 37-47, the host cell of any one of Embodiments 48-54, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and / or the composition of any one of Embodiments 55-58, for use the manufacture of a medicament for treating a disease or disorder associated with a KRAS G12V or a NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and, wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

[0148] The present disclosure also provides the following non-limiting Embodiments:

[0149] Embodiment la. A binding protein that is capable of binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing 2 or 3 methyl groups.

[0150] Embodiment 2a. The binding protein of Embodiment la, wherein the N-terminal lysine of the peptide comprises a side chain bearing 2 methyl groups.

[0151] Embodiment 3 a. The binding protein of Embodiment la, wherein the N-terminal lysine of the peptide comprises a side chain bearing 3 methyl groups.

[0152] Embodiment 4a. The binding protein of any one of Embodiments la-3 a, wherein the binding protein is also capable of binding to peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing 0 or 1 methyl groups.

[0153] Embodiment 5a. The binding protein of any one of Embodiments la-4a, wherein the binding protein comprises a binding domain that binds the peptide:HLA complex, wherein the binding domain optionally comprises an immunoglobulin superfamily variable domain.

[0154] Embodiment 6a. The binding protein of Embodiment 5a, wherein binding domain comprises a TCR a-chain variable domain (Va) and / or a TCR P-chain variable domain (VP), wherein the binding domain optionally comprises the Va domain and the VP domain.

[0155] Embodiment 7a. The binding protein of any one of Embodiments la-9a, comprising a heavy chain variable domain (VH) and / or a light chain variable domain (VL) of a TCR-mimic antibody, wherein the binding domain optionally comprises the VH domain and the VL domain.

[0156] Embodiment 8a. A binding protein comprising a TCR a-chain variable domain (Va) and a TCR P-chain variable domain (VP), wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV, the N-terminal lysine of the peptide bears zero, one, two, or three methyl groups, and the HLA is HLA-A*02:01, and wherein the Va domain comprises complementarity determining region (CDR)la, CDR2a, and CDR3a amino acid sequences and the VP domain comprises CDRip, CDR2P, and CDR3P amino acid sequences, wherein the CDRla, CDR2a, CDR3a CDRip, CDR2P, and CDR3P amino acid sequences are: (i) DSSSTY, IFSNMDM, CAEIEDRTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRT, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRT, as shown in Table Z; (ii) DSSSTY, IFSNMDM, CAEIEDSTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDST, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDST, as shown in Table Z; (iii) DSSSTY, IFSNMDM, CAEIEDRSDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRS, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRS, as shown in Table Z; (iv) DSSSTY, IFSNMDM, CAEITGNTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR TGNT, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR TGNT, as shown in Table Z; (v) DSSSTY, IFSNMDM, CAEIEDVADKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDVA, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR EDVA, as shown in Table Z; (vi) DSSSTY, IFSNMDM, CAEIEARRDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EARR, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EARR, as shown in Table Z; (vii) YGATPY, YFSGDTLV, CAVGADSNYQLIW, SGHNS, FNNNVP, and CASSLVFYNEQFF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-l, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-l, as shown in Table Z; (viii) NSAFQY, TYSSGN, CAMSDDWGKLQF, PRHDT, FYEKMQ, and CASSTIAGGRGNEQFF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-2, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-2, as shown in Table Z; or (ix) DSAIYN, IQSSQRE, CAVPPGDDYKLSF, MDHEN, SYDVKM, and CASSPIQRGDQPQHF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-4, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR A2UoMl-4, as shown in Table Z.

[0157] Embodiment 10a. A T cell receptor comprising an alpha chain and a beta chain, wherein: The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDRT (4UM), as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDRT (4UM), as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRT, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR EDRT, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDST, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDST, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDST, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR EDST, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDRS, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDRS, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRS, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR EDRS, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR TGNT, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR TGNT, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR TGNT, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR TGNT, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDVA, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDVA, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDVA, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDVA, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EARR, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EARR, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EARR, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EARR, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-l, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-l, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-l, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-l, as shown in Table Z; The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-2, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-2, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-2, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-2, as shown in Table Z; or The alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-4, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-4, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-4, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-4, as shown in Table Z.

[0158] Embodiment I la. A T cell receptor comprising an alpha chain and a beta chain, wherein: The alpha chain comprises the alpha chain variable domain TCR EDRT (4UM), as shown in Table Y, and the beta chain variable domain of TCR EDRT (4UM), as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EDST, as shown in Table Y, and the beta chain variable domain of TCR EDST, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EDRS, as shown in Table Y, and the beta chain variable domain of TCR EDRS (4UM) or of TCR EDRS, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR TGNT, as shown in Table Y, and the beta chain variable domain of TCR TGNT, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EDVA, as shown in Table Y, and the beta chain variable domain of TCR EDVA, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR EARR, as shown in Table Y, and the beta chain variable domain of TCR EARR, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR A2UoM-l-l, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-l, as shown in Table Z; The alpha chain comprises the alpha chain variable domain TCR A2UoM-l-2, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-2, as shown in Table Z; or The alpha chain comprises the alpha chain variable domain TCR A2UoM-l-4, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-4, as shown in Table Z.

[0159] Embodiment 12a. A binding protein comprising a TCR alpha chain variable domain of any one of the TCRs shown in Table Y and the corresponding beta chain variable domain shown in Table X.

[0160] Embodiment 13 a. The binding protein of any one of Embodiments la- 12a, further comprising a TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP), optionally comprising a Ca and a C .

[0161] Embodiment 14a. The binding protein of Embodiment 12a or 13a or the T cell receptor of Embodiment 10a or 1 la, wherein the Ca comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, one of the following amino acid sequences:

[0162] IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS;

[0163] IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKCVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS;

[0164] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKV AGFNLLMTLRLWSS .

[0165] Embodiment 15a. The binding protein of Embodiment 12a, 13a, or 14a or T cell receptor of Embodiment 10a, I la, or 14a, wherein the CP comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, one of the following the amino acid sequences:

[0166] DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE

[0167] VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ

[0168] DRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDF;

[0169] DLNKVFPPEV AVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDP QPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE AWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF;

[0170] DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE

[0171] VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ

[0172] DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG;

[0173] DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE

[0174] VHSGVCTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG.

[0175] Embodiment 16a. The binding protein or T cell receptor of any one of Embodiments 10a- 15a, wherein the Ca and the Cp comprise or consist of an engineered variant of a human Ca and an engineered variant of a human CP, respectively.

[0176] Embodiment 17a. The binding protein or T cell receptor of any one of Embodiments 10a- 16a, wherein the Ca, the CP, or both comprise modification(s) that promote preferential pairing of the Ca to the Cp.

[0177] Embodiment 18a. The binding protein or T cell receptor of any one of Embodiments 10a- 16a, wherein the Ca and the CP each comprises an introduced cysteine residue that promotes preferential pairing of the Ca to the Cp.

[0178] Embodiment 19a. The binding protein or T cell receptor of any one of Embodiments 10a- 16a, wherein the Ca comprises a T48C substitution and the Cp comprises a S57C substitution to promote preferential pairing of the Ca to the Cp.

[0179] Embodiment 20a. The binding protein or T cell receptor of any one of Embodiments la- 19a, consisting essentially of a TCR a chain and a TCR P chain. Embodiment 21a. The binding protein or T cell receptor of any one of Embodiments la-20a, wherein the binding protein comprises a TCR, a single-chain TCR (scTCR), a singlechain T cell receptor variable fragment (scTv), or a chimeric antigen receptor (CAR).

[0180] Embodiment 22a. The binding protein of Embodiment 21, wherein the binding protein comprises a TCR.

[0181] Embodiment 23 a. The binding protein of any one of Embodiments 1-22, wherein the binding protein comprises an EC50 of at most 100 nM, at most 50 nM, at most 25 nM, at most 10 nM, at most 1 nM, at most 750 pM, at most 500 pM, at most 250 pM, at most 100 pM, at most 75 pM, or at most 60 pM in a CD 137 surface expression assay for functional avidity to the peptide.

[0182] Embodiment 24a. An isolated polynucleotide encoding the binding protein or T cell receptor of any one of Embodiments la-23 a.

[0183] Embodiment 25a. An isolated polynucleotide enoding a Va domain, a VP domain, a TCR a-chain, and / or a TCR P-chain of the binding protein or T cell receptor of any one of Embodiments 1-23.

[0184] Embodiment 26a. The polynucleotide of Embodiment 24a or 25a, further comprising: (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 coreceptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii)a polynucleotide of (i) and a polynucleotide of (ii).

[0185] Embodiment 27a. The polynucleotide of Embodiment 26a, comprising:

[0186] (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co- receptor a chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor p chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).

[0187] Embodiment 28a. The polynucleotide of Embodiment 26a or 27a, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between: (1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; and / or (2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain.

[0188] Embodiment 29a. The polynucleotide of any one of Embodiments 26a-28a, comprising, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnBP); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCPi)-(pnCD8a)- (pnSCP2)-(pnCD8P); (iv) (pnBP)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnCD8a); (v) (pnCD8a)- (pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8P); or (vi) (pnCD8P)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnBP is the polynucleotide encoding a binding protein, and wherein pnSCPi and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0189] Embodiment 30a. The polynucleotide of any one of Embodiments 26a-29a, wherein the encoded binding protein comprises a TCRa chain and a TCRP chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.

[0190] Embodiment 31a. The polynucleotide of Embodiment 30a, comprising, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa); (iii) (pnCD8a)- (pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (iv) (pnCD8P)-(pnSCPi)- (pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (v) (pnTCRP)-(pnSCPi)-(pnTCRa)- (pnSCP2)-(pnCD8a)-(pnSCP3)-(pnCD8P); (vi) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)- (pnCD8P)-(pnSCP3)-(pnCD8a); (vii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8a)- (pnSCP3)-(pnCD8P); (viii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8P)-(pnSCP3)- (pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnTCRa is the polynucleotide encoding a TCR a chain, wherein pnTCRP is the polynucleotide encoding a TCR P chain, and wherein pnSCPi, pnSCP2, and pnSCPs are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0191] Embodiment 32a. The polynucleotide of Embodiment 31a, wherein the pnSCPl encodes a T2A peptide, the pnSCP2 encodes a P2A peptide, and the pnSCP3 encodes a P2A peptide.

[0192] Embodiment 33 a. A polynucleotide encoding any one of the following amino acid sequences:

[0193] AGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPID DSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLVFYNEQFFGPGTRLTVLEDL KNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSR GGSGATNFSLLKQAGDVEENPGPMLLELIPLLGIHFVLRTARAQSVTQPDIHITVSEGASL ELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRK PSVHWSDAAEYFCAVGADSNYQLIWGAGTKLIIKPDIQNPDPARH;

[0194] MLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQ FLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSTIAGGRGNEQF FGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG KEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGPMMKSLRVLLVILWLQLSWVWS QQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKE DGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDDWGKLQFGAGTQVVVTPDIQNPD PARH;

[0195] VI<VTQSSRYLVI<RTGEI<VFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVI< MKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPIQRGDQPQHFGDGTRLSIL EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DFGSGATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEG ENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYI AASQPGDSATYLCAVPPGDDYKLSFGAGTTVTVRANIQNPDPARH.

[0196] Embodiment 34a. A polynucleotide encoding an amino acid sequence comprising AGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPID DSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLVFYNEQFFGPGTRLTVLEDL KNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSR GGSGATNFSLLKQAGDVEENPGPMLLELIPLLGIHFVLRTARAQSVTQPDIHITVSEGASL ELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRK PSVHWSDAAEYFCAVGADSNYQLIWGAGTKLIIKPDIQNPDPARH.

[0197] Embodiment 35a. A polynucleotide encoding an amino acid sequence comprising MLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISF YEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSTIAGGRGNEQFFGPG TRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVH SGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMA MVKRKDSRGGSGATNFSLLKQAGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEV EQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFT AQVDKSSKYISLFIRDSQPSDSATYLCAMSDDWGKLQFGAGTQVVVTPDIQNPDPARH.

[0198] Embodiment 36a. A polynucleotide encoding an amino acid sequence comprising VKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKG DIPEGYS VSREKKERF SLILES ASTNQTSMYLC AS SPIQRGDQPQHFGDGTRLSILEDLNK VFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSG ATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVL NCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQP GDSATYLCAVPPGDDYKLSFGAGTTVTVRANIQNPDPARH.

[0199] Embodiment 37a. An expression vector, comprising a polynucleotide of any one of

[0200] Embodiments 24a-36a operably linked to an expression control sequence.

[0201] Embodiment 38a. The expression vector of Embodiment 37a, wherein the expression control sequence comprises an MSCV promoter.

[0202] Embodiment 39a. The expression vector of Embodiment 37a or Embodiment 38a, wherein the expression control sequence drives expression of a single mRNA encoding an extracellular portion of a CD8 co-receptor a chain, an extracellular portion of an CD8 coreceptor P chain, the TCR a chain, and the TCR P chain.

[0203] Embodiment 40a. The expression vector of any one of Embodiments 37a-39a, wherein the vector is capable of delivering the polynucleotide to a host cell.

[0204] Embodiment 41a. The expression vector of Embodiment 40a, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

[0205] Embodiment 42a. The expression vector of Embodiment 41a, wherein the human immune system cell is a CD4+T cell, a CD8+T cell, a CD4 CD8' double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof.

[0206] Embodiment 43a. The expression vector of Embodiment 42a, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0207] Embodiment 44a. The expression vector of any one of Embodiments 37a-43a, wherein the vector is a viral vector.

[0208] Embodiment 45a. The expression vector of Embodiment 44a, wherein the viral vector is a lentiviral vector or a y-retroviral vector.

[0209] Embodiment 46a. The expression vector of Embodiment 44a, wherein the viral vector is a self-inactivating lentiviral vector.

[0210] Embodiment 47a. The expression vector of Embodiment 44a or Embodiment 46a, wherein the viral vector is a third generation lentiviral vector.

[0211] Embodiment 48a. A host cell modified to comprise the polynucleotide of any one of Embodiments 24a-36a and / or the expression vector of any one of Embodiments 37a-47a and / or to express the binding protein of any one of Embodiments la-23 a.

[0212] Embodiment 49a. The host cell of Embodiment 48a, wherein the modified cell comprises a hematopoietic progenitor cell and / or a human immune cell.

[0213] Embodiment 50a. The host cell of Embodiment 49a, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.

[0214] Embodiment 51a. The host cell of Embodiment 50a, wherein the immune cell comprises a CD4+T cell, a CD8+T cell, a CD4' CD8' double negative T cell, a y5 T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4+T cell and a CD8+T cell, wherein, further optionally, the CD4+T cell, the CD8+T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

[0215] Embodiment 52a. The host cell of any one of Embodiments 48a-51a, wherein the modified cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.

[0216] Embodiment 53a. The host cell of Embodiment 52a, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.

[0217] Embodiment 54a. The host cell of Embodiment 52a or 53a, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.

[0218] Embodiment 55a. A composition comprising the host cell of any one of Embodiments 48a-54a and a pharmaceutically acceptable carrier, diluent, or excipient.

[0219] Embodiment 56a. The composition of Embodiment 55a, comprising at least about 30% modified CD4+T cells, combined with (ii) a composition comprising at least about 30% modified CD8+T cells, in about a 1 : 1 ratio.

[0220] Embodiment 57a. The composition of Embodiment 55a or 56a, wherein the composition contains substantially no naive T cells.

[0221] Embodiment 58a. A composition comprising: (i) the binding protein of any one of Embodiments la-23 a; (ii) the polynucleotide of any one of Embodiments 24a-36a;

[0222] (iii) the expression vector of any one of Embodiments 37a-47a; and / or (iv)the host cell of any one of Embodiments 48a-54a, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0223] Embodiment 59a. A method for treating a disease or disorder associated with a KRAS G12V mutation or a NRAS G12V mutation or a HRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of: (i)the binding protein of any one of Embodiments la-23 a; (ii) the polynucleotide of any one of Embodiments 24a-36a; (iii) the expression vector of any one of Embodiments 37a-47a; (iv) the host cell of any one of Embodiments 48a-54a, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and wherein, optionally, the host cell is autologous, allogeneic, or syngeneic to the subject; and / or (v) the composition of any one of Embodiments 55a-58a, wherein, optionally, the method further comprises administering to the subject, or the subject has received, an effective amount of an agent that inhibits expression or activity of SPT6, wherein, further optionally, the agent that inhibits expression or activity of SPT6 comprises an siRNA, e.g., as available from Creative Biolabs (e.g., at creative-biolabs.com / gene-therapy / small-interfering-rna-sirna-anti- human-supt6h-42027.htm) or from Origene (e.g.. at origene.com / catalog / rnai / sirna-oligo- duplexes / sr304661 / spt6-supt6h-human-sima-oligo-duplex-locus-id-6830).

[0224] Embodiment 60a. The method of Embodiment 59a, wherein the disease or disorder comprises a cancer, wherein the cancer is optionally a solid cancer or a hematological malignancy.

[0225] Embodiment 61a. The method of Embodiment 59a or 60a, wherein the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a nonsmall-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

[0226] Embodiment 62a. The method of any one of Embodiments 59a-61a, wherein the binding protein, polynucleotide, vector, host cell, or composition is administered to the subject parenterally or intravenously.

[0227] Embodiment 63a. The method of any one of Embodiments 59a-62a, wherein the method comprises administering a plurality of doses of any one or more of (i)-(v) to the subject.

[0228] Embodiment 64a. The method of Embodiment 63 a, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.

[0229] Embodiment 65a. The method of any one of Embodiments 59a-64a, wherein the composition comprises the host cell or the composition comprising the host cell, and wherein the method comprises administering the host cell or composition to the subject at a dose of about 104cells / kg to about 1011cells / kg.

[0230] Embodiment 66a. The method of any one of Embodiments 59a-65a, wherein the method comprises administering to the subject at least 5xlOA8, at least lxlOA9, at least 5xlOA9, at least lxl0A10, at least 1.5xl0A10, at least 2xl0A10, or at least 5xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

[0231] Embodiment 67a. The method of any one of Embodiments 59a-65a, wherein the method comprises administering to the subject at most 5xlOA9, at most lxl0A10, at most 1.5xl0A10, at most 2x10A10, at most 5xl0A10, at most lxlOAl l, or at most 5xl0Al l viable host cells that comprise the binding protein, optionally in a single dose.

[0232] Embodiment 68a. The method of any one of Embodiments 59a-65a, wherein the method comprises administering to the subject about 5xlOA9, about 6xlOA9, about 7xlOA9, about 8xlOA9, about 9xlOA9, about lxl0A10, about l. lxl0A10, about 1.2xl0A10, about 1.3xl0A10, about 1.4xl0A10, about 1.5xl0A10, about 1.6xl0A10, about 1.7xl0A10, about 1.8xl0A10, about 1.9xl0A10, or about 2xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

[0233] Embodiment 69a. The method of any one of Embodiments 59a-65a, wherein the method comprises administering to the subject about 5 xlOA9 to about 1 xlOAl 1, about 5 xlOA9 to about 5 xl0A10, about 5 xlOA9 to about 2 xl0A10, about 5 xlOA9 to about 1.5 xl0A10, about 5 xlOA9 to about 1 xl0A10, about 1 xl0A10 to about 1 xlOAl l, about 1 xl0A10 to about 5 xl0A10, about 1 xl0A10 to about 2 xl0A10, or about 1 xl0A10 to about 1.5 xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

[0234] Embodiment 70a. The method of any one of Embodiments 59a-69a, further comprising determining that the subject expresses HLA-A*02:01, prior to administering the binding protein, TCR, polynucleotide, vector, host cell, or composition, and / or determining that the subject expresses a KRAS G12V 5-14, NRAS G12V 5-14, or HRAS G12V 5-14 peptide comprising one or more methyl groups at a N-terminal lysine of the peptide prior to administering the binding protein, TCR, polynucleotide, vector, host cell, or composition.

[0235] Embodiment 71a. The method of any one of Embodiments 59a-70a, wherein the method further comprises administering a cytokine to the subject.

[0236] Embodiment 72a. The method of Embodiment 71a, wherein the cytokine comprises

[0237] IL-2, IL-15, or IL-21.

[0238] Embodiment 73a. The method of any one of Embodiments 59a-72a, wherein the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

[0239] Embodiment 74a. The binding protein of any one of Embodiments la-23 a, the polynucleotide of any one of Embodiments 24a-36a, the expression vector of any one of Embodiments 37a-47a, the host cell of any one of Embodiments 48a-54a, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and / or the composition of any one of Embodiments 55a-58a, for use in a method for treating a disease or disorder associated with a KRAS G12V or a NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

[0240] Embodiment 75a. The binding protein of any one of Embodiments la-23a, the polynucleotide of any one of Embodiments 24a-36a, the expression vector of any one of Embodiments 37a-47a, the host cell of any one of Embodiments 48a-54a, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and / or the composition of any one of Embodiments 55a-58a, for use the manufacture of a medicament for treating a disease or disorder associated with a KRAS G12V or a NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and, wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

[0241] Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

[0242] It will be understood that certain exemplified binding proteins described herein may be referred to using alternative naming. For example, TCR “EDRT” may also identified as TCR “4UM”, TCR “EDRT (4UM)”, and “TCR2 EDRT”. For example, TCR “EDST” may also identified as “TCR2 EDST”. For example, TCR “EDRS” may also identified as “TCR2 EDRS”. For example, TCR “TGNT” may also identified as “TCR2 TGNT”. For example, TCR “EDVA” may also identified as “TCR2 EDVA”. For example, TCR “EARR” may also identified as “TCR2 EARR”.

[0243] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term “about” means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms “include”, “have”, and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

[0244] In addition, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure.

[0245] The term “consisting essentially of’ is not equivalent to “comprising” and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) “consists essentially of’ a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carb oxy -terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% ) of the length of a domain, region, module, or protein and do not substantially affect (ie., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% ) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity or avidity of a binding protein).

[0246] As used herein, “protein” or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers. In some embodiments, a “peptide” (e.g., a peptide antigen) refers to a polymer of about 8-10 amino acid residues in length.

[0247] It will be appreciated that a multimeric protein or protein complex (e.g., a TCR comprising an alpha chain polypeptide and a beta chain polypeptide and sharing one or more disulfide bonds) may still be referred to as a “protein” or “polypeptide”.

[0248] As used herein, a “hematopoietic progenitor cell” is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with a CD24LOLin CD117+phenotype or those found in the thymus (referred to as progenitor thymocytes).

[0249] As used herein, an “immune system cell” or “immune cell” means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+T cell, a CD8+T cell, a CD4' CD8' double negative T cell, a y5 T cell, a regulatory T cell, a natural killer cell, a natural killer T cell, and a dendritic cell. Macrophages and dendritic cells can be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

[0250] A “T cell” or “T lymphocyte” is an immune system cell that matures in the thymus and produces a T cell receptor (TCR). TCRs are known in the art and described herein. A T cell in which endogenous TCR expression has been abrogated by, for example, disruption of one or more TCR genes, may, nonetheless, be referred to as a T cell. T cells can be naive (“TN”; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased or no expression of CD45RO as compared to TCM(described herein)), memory T cells (TM) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced, cytotoxic). TMcan be further divided into subsets of central memory T cells (TCMexpresses CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEMexpress CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TE) refers to antigen-experienced CD8+cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to TCM, and are positive for granzyme and perforin (e.g., upon stimulation). Helper T cells (TH) are CD4+cells that influence the activity of other immune cells by releasing cytokines. CD4+T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of, e.g., transcription factors, and other cells and signals. T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4+CD25+(Foxp3+) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8+CD28‘, and Qa-1 restricted T cells.

[0251] As used herein, "enriched" or "depleted" with respect to amounts of cell types in a mixture refers to an increase in the number of the "enriched" type, a decrease in the number of the "depleted" cells, or both, in a mixture of cells resulting from one or more enriching or depleting processes or steps. Thus, depending upon the source of an original population of cells subjected to an enriching process, a mixture or composition may contain 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65%, or more 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more (in number or count) of the "enriched" cells. Cells subjected to a depleting process can result in a mixture or composition containing 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5%v, 4% or less, 3% or less, 2% or less, or 1% percent or less (in number or count) of the "depleted" cells. In certain embodiments, amounts of a certain cell type in a mixture will be enriched and amounts of a different cell type will be depleted, such as enriching for CD4+cells while depleting CD8+cells, or enriching for CD62L+cells while depleting CD62L cells, or combinations thereof.

[0252] “T cell receptor” (TCR) refers to an immunoglobulin superfamily member having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally comprises a heterodimer having a and P chains (also known as TCR a and TCRP, respectively), or y and 5 chains (also known as TCRy and TCR5, respectively). In certain embodiments, a polynucleotide encoding a binding protein of this disclosure, e.g., a TCR, can be codon optimized to enhance expression in a particular host cell, such, for example, as a cell of the immune system, a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et al., Clin. Immunol. 119: 135, 2006). Exemplary T cells that can express binding proteins and TCRs of this disclosure include CD4+T cells, CD8+T cells, and related subpopulations thereof (e.g., naive, central memory, stem cell memory, effector memory).

[0253] Like immunoglobulins (e.g., antibodies), the extracellular portion of TCR chains (e.g., a- chain, P-chain) can contain two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or VaP-chain variable domain or Vp at the N-terminus, and one constant domain (e.g., a-chain constant domain or Ca,) adjacent the cell membrane. Also, like immunoglobulins, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit, or other mammal. In preferred embodiments, a TCR comprises six CDRs from a human TCR, or comprises six CDRs, one or more of which is engineered from a human TCR CDR sequence. In some embodiments, a TCR comprises variable domains from a human TCR, or comprises one or two variable domains engineered from human TCR variable domains to comprise one, two, or three amino acid substitutions relative to a human TCR variable domain.

[0254] The term “variable region” or “variable domain” refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR a-chain or P-chain (or y chain and 5 chain for y5 TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., TCR) to antigen. The variable domains of the a-chain and P-chain (Va and VP, respectively) of a native TCR generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs. The Va domain is encoded by two separate DNA segments, the variable gene segment, and the joining gene segment (V-J); the VP domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J). A single Va or VP domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind a particular antigen may be isolated using a Va or VP domain from a TCR that binds the antigen to screen a library of complementary Va or VP domains, respectively.

[0255] The terms “complementarity determining region,” and “CDR,” are synonymous with “hypervariable region” or “HVR,” and are known in the art to refer to sequences of amino acids within immunoglobulin (e.g., TCR) variable regions. CDRs confer antigen specificity and binding affinity and are separated from one another in primary amino acid sequence by framework regions. In general, there are three CDRs in each TCR a-chain variable region (aCDRl, aCDR2, aCDR3 (also identified as CDRla, CDR2a, and CDR3a, respectively)) and three CDRs in each TCR P-chain variable region (PCDR1, PCDR2, PCDR3 (also identified as CDRip, CDR2P, and CDR3P, respectively)). In TCRs, CDR3 is thought to be the main CDR responsible for recognizing processed antigen. In general, CDR1 and CDR2 interact mainly or exclusively with the MHC.

[0256] CDR1 and CDR2 are encoded within the variable gene segment of a TCR variable region-coding sequence, whereas CDR3 is encoded by the region spanning the variable and joining segments for Va, or the region spanning variable, diversity, and joining segments for Vp. Thus, if the identity of the variable gene segment of a Va or VP is known, the sequences of their corresponding CDR1 and CDR2 can be deduced; e.g., according to a numbering scheme as described herein. Compared with CDR1 and CDR2, CDR3, and in particular CDR3P, is typically significantly more diverse due to the addition and loss of nucleotides during the recombination process.

[0257] TCR variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), allowing equivalent residue positions to be annotated and for different molecules to be compared using, for example, ANARCI software tool (2016, Bioinformatics 15:298-300). A numbering scheme provides a standardized delineation of framework regions and CDRs in the TCR variable domains. In certain embodiments, a CDR of the present disclosure is identified or defined according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 2T.55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, a CDR (e.g., CDR3) is identified or defined in accordance with the IMGT- junction definition (including CYS 104 and PHE 118 or TRP 118). In some embodiments, a CDR (e.g., CDR3, or all six CDRs of a binding protein) is identified or defined in accordance with the IMGT definition (or scheme or method) (rearranged CDR3 corresponding to IMGT positions 105-117 within the variable domain). In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the Kabat numbering scheme or method. In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the Chothia numbering scheme or method. In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the EU numbering scheme or method. In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined according to the enhanced Chothia numbering scheme or method. In some embodiments, a CDR (or all six CDRs of a binding protein) of the present disclosure is identified or defined of the present disclosure is identified according to the Aho numbering scheme or method.

[0258] The source of a TCR as used in the present disclosure may be from any of a variety of animal species, such as a human, mouse, rat, rabbit, or other mammal. TCR constant domain sequences may be from, for example, human, mouse, marsupial e.g, opossum, bandicoot, wallaby), shark, or non-human primate. In certain preferred embodiments, TCR constant domain sequences are human or comprise engineered variants of human sequences. TCR constant domains may be engineered to, for example, improve pairing, expression, stability, or any combination of these. See, e.g., Cohen et al., Cancer Res, 2007; Kuball et al., Blood 2007; and Haga-Friedman et al., Journal of Immunology 2009. Examples of engineering in TCR Ca and CP include mutation of a native amino acid to a cysteine so that a disulfide bond forms between the introduced cysteine of one TCR constant domain and a native cysteine of the other TCR constant domain. Such mutations can include, e.g., T48C in Ca, T57C or S57C in CP, or both. Also provided are embodiments wherein cognate TCR constant domains comprise mutations so that, for example, one TCR constant domain (e.g., one of Ca and CP) comprises an introduced so-called “cavity” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having smaller side chains) and the other (e.g., the other of Ca and CP) comprises a compensatory so-called “protuberance” (e.g., obtainable by replacing one or more native amino acid with one or more amino acids having larger side chains), similar to a “knob-into-hole” configuration used to promote preferential pairing of antibody heavy chains. Also provided are embodiments wherein TCR constant domain amino acids are mutated to introduce or modify charge properties so as to favor pairing of the mutated constant domains. Examples of mutations that may be made in Ca and CP to promote specific pairing by a knobs-into-holes-type mechanism or by a charge-pairing mechanism are provided in Voss et al., J. Immunol 750(l):391-401 (2008) doi.org / 10.4049 / jimmunol.180.1.391; see also US Patent No.

[0259] 9,062,127. The TCR constant domain mutations, mutated TCR constant domains, and methods used to identify sites for mutation, described in these documents, are incorporated herein by reference.

[0260] Mutations to improve stability can include a mutation in the Ca transmembrane domain from the sequence LSVIGF to the sequence LLVIVL (“L-V-L” mutation; see Haga-Friedman et al., J Immunol 755:5538-5546 (2012), the TCR mutations and mutant TCR constant domain sequences of which are incorporated herein by reference).

[0261] Engineered proteins comprising TCR features include scTCRs, scTvs, TCR-CARs, disulfide-stabilized TCR variable fragments (dsTvs; see, e.g., Reiter et al., Immunity 2()3)281- 287 (1995), doi: 10.1016 / 1074-7613(95)90052-7), and multispecific T cell engager proteins comprising any of the foregoing.

[0262] As used herein, the term “CD8 co-receptor” or “CD8” means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 co-receptor assists in the function of cytotoxic T cells (CD8+) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21 :630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1 :81-88, 2004). There are five (5) human CD8 beta chain isoforms (see UniProtKB identifier Pl 0966) and a single human CD8 alpha chain isoform (see UniProtKB identifier P01732). In some embodiments, a CD8 beta chain can be an Ml isoform. “CD4” is an immunoglobulin co-receptor glycoprotein that assists the TCR of CD4+ cells in communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002)). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (DI to D4) that are expressed at the cell surface. During antigen presentation, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds MHCII al / pi). Without wishing to be bound by theory, it is believed that close proximity to the TCR complex allows CD4-associated kinase molecules to phosphorylate the immunoreceptor tyrosine activation motifs (IT AMs) present on the cytoplasmic domains of CD3. This activity is thought to amplify the signal generated by the activated TCR in order to produce or recruit various types of immune system cells, including T helper cells, and immune responses.

[0263] In certain embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with a CD3 complex. “CD3” is a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999) that is associated with antigen signaling in T cells. In mammals, the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3(^ chains. The CD3y, CD3P, and CD3s chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD3P, and CD3s chains are negatively charged, which is believed to allow these chains to associate with positively charged regions of T cell receptor chains. The intracellular tails of the CD3y, CD3P, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine based activation motif or IT AM, whereas each CD3(^ chain has three. Without wishing to be bound by theory, it is believed that the IT AMs are important for the signaling capacity of a TCR complex. CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.

[0264] As used herein, “TCR complex” refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3(^ chains, a TCRa chain, and a TCRP chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3(^ chains, a TCRy chain, and a TCRP chain.

[0265] A “component of a TCR complex”, as used herein, refers to a TCR chain (i.e., TCRa, TCRP, TCRy or TCR5), a CD3 chain (i.e., CD3y, CD35, CD3s or CD3Q, or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRP, CD3y, CD35, and two CD3s chains).

[0266] As used herein, "fusion protein" or "fusion polypeptide" refers to a protein that, in a single chain, has at least two distinct domains and / or motifs, wherein the domains or motifs are not naturally found together (e.g., in the given arrangement, order, or number, or at all) in a protein. In certain embodiments, a fusion protein comprises at least two distinct domains and / or motifs that are not found together in a single naturally occurring peptide or polypeptide. In certain embodiments, a fusion protein comprises amino acid sequences from two or more distinct polypeptides. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized. A fusion protein may further contain other components, such as a tag, a linker, or a transduction marker. In certain embodiments, a fusion protein expressed or produced by a host cell (e.g., a T cell) locates to the cell surface, where the fusion protein can be anchored to or disposed at least partially within the cell membrane.

[0267] “Chimeric antigen receptor” (CAR) refers to a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences, domains, or motifs, linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs can include an extracellular portion comprising an antigen-binding domain (e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or obtained from a TCR specific for a cancer antigen, a scFv derived or obtained from an antibody, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3) 220 (2016), Stone et al., Cancer Immunol. Immunother., 63(11): 1163 (2014), and Walseng et al., Scientific Reports 7: 10713 (2017), which CAR constructs and methods of making the same are incorporated by reference herein). CARs of the present disclosure that specifically bind to a Ras antigen (e.g., in the context of a peptide:HLA complex) comprise a TCR Va domain and a VP domain.

[0268] Any polypeptide of this disclosure can, as encoded by a polynucleotide sequence, comprise a “signal peptide” (also known as a leader sequence, leader peptide, or transit peptide). Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell (e.g., to be inserted into or localize to a cell membrane, or to be secreted by the cell, or to be contained within the cell). In some contexts, signal peptides are from about 15 to about 22 amino acids in length. Non-limiting examples of signal peptides include: a signal peptide native to a mammalian (e.g., human) TCRa or TCRP chain; MLLLVTSLLLCELPHPAFLLIP (from GM-CSF); the signal peptide MALPVTALLLPLALLLHAARP (from CD8a); the signal peptide MRPRLWLLLAAQLTVLHGNSV (from CD8P); and the signal peptide METDTLLLWVLLLWVPGSTG (from murine IgG kappa light chain). It will be appreciated that any suitable naturally occurring or engineered signal peptide can be employed. Certain signal peptides and characteristics of these are decribed in Owji et al., European Journal of Cell Biology 97(6):422-441 (2018), and in Ling et al. Front. Immunol. (2020) doi.org / 10.3389 / fimmu.2020.604318; the signal peptides of which are incorporated herein by reference. A signal peptide may be removed (in whole or in part) from the polypeptide during or once localization (e.g., membrane insertion) or secretion is completed. In some embodiments, a signal peptide is completely removed from the polypeptide. In some embodiments, less than all, but typically no more than one, two, three, four, five, or six amino acids of the signal peptide remain with the polypeptide and the rest of the signal peptide is removed. Polypeptides that have a signal peptide are referred to herein as a “pre-protein” and polypeptides having their signal peptide removed are referred to herein as “mature” proteins or polypeptides. In any of the herein disclosed embodiments, a binding protein or fusion protein comprises, or is, a mature protein, or is or comprises a pre-protein.

[0269] A “linker” refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex). In certain embodiments, a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids. Exemplary linkers include glycine-serine linkers.

[0270] “Antigen” or “Ag” as used herein refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells (e.g., T cells), or both. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen, or that endogenously (e.g., without modification or genetic engineering by human intervention) express a mutation or polymorphism that is immunogenic.

[0271] A “neoantigen,” as used herein, refers to a host cellular product containing a structural change, alteration, or mutation that creates a new antigen or antigenic epitope that has not previously been observed in the subject’s genome (z.e., in a sample of healthy tissue from the subject) or been “seen” or recognized by the host's immune system, which: (a) is processed by the cell’s antigen-processing and transport mechanisms and presented on the cell surface in association with an MHC (e.g., HLA) molecule; and (b) elicits an immune response (e.g., a cellular (T cell) response). Neoantigens may originate, for example, from coding polynucleotides having alterations (substitution, addition, deletion) that result in an altered or mutated product, or from the insertion of an exogenous nucleic acid molecule or protein into a cell, or from exposure to environmental factors (e.g., chemical, radiological) resulting in a genetic change. Neoantigens may arise separately from a tumor antigen, or may arise from or be associated with a tumor antigen. “Tumor neoantigen” (or “tumor-specific neoantigen”) refers to a protein comprising a neoantigenic determinant associated with, arising from, or arising within a tumor cell or plurality of cells within a tumor. Tumor neoantigenic determinants are found on, for example, antigenic tumor proteins or peptides that contain one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g., pancreas cancer, lung cancer, colorectal cancers), as well as proteins or peptides from viral open reading frames associated with virus-associated tumors (e.g., cervical cancers, some head and neck cancers). The terms “antigen” and “neoantigen” are used interchangeably herein when referring to a Ras antigen comprising a mutation as disclosed herein.

[0272] The term “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0273] As used herein, the term “KRAS (or NRAS or HRAS) antigen (or neoantigen)” or “KRAS (or NRAS or HRAS) peptide antigen (or neoantigen)” or “KRAS (NRAS or HRAS) peptide” refers to a naturally or synthetically produced peptide portion of a KRAS or NRAS or HRAS protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids, up to about 20 amino acids, and comprising at least one amino acid alteration caused by a G12 (e.g., G12V) mutation (wherein position 12 is in reference to the full-length KRAS protein sequence set forth in SEQ ID NO: 1; and is also in reference to the full- length NRAS and HRAS protein sequence set forth in SEQ ID NOs: 3 and 4, respectively), which peptide can form a complex with a MHC (e.g., HLA) molecule, and a binding protein of this disclosure specific for a KRAS or NRAS or HRAS peptide:MHC (e.g., HLA) complex can specifically bind to such as complex. An exemplary KRAS (or NRAS or HRAS) antigen comprises, consists essentially of, or consists of a peptide having the amino acid sequence of SEQ ID NO: 2. Other exemplary KRAS (or NRAS or HRAS) antigen comprise, consist essentially of, or consist of a peptide having the amino acid sequence of any one of SEQ ID NOs: 2, 5, 6, or 7.

[0274] “Major histocompatibility complex” (MHC) refers to glycoproteins that deliver peptide antigens to a cell surface of all nucleated cells. MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non-covalently associated P2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and P, both of which span the membrane. Each chain comprises two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8+T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4+T cells. Human MHC is referred to as human leukocyte antigen (HLA). HL As corresponding to “class I” MHC present peptides from inside the cell and include, for example, HLA-A, HLA-B, and HLA-C. Alleles include, for example, HLA A*02:01. HLAs corresponding to “class II” MHC present peptides from outside the cell and include, for example, HLA-DP, HLA-DM, HLA-DOA, HLA- DOB, HLA-DQ, and HLA-DR.

[0275] Principles of antigen processing by antigen presenting cells (APC) (such as dendritic cells, macrophages, lymphocytes or other cell types), and of antigen presentation by APC to T cells, including major histocompatibility complex (MHC)-restricted presentation between immunocompatible (e.g., sharing at least one allelic form of an MHC gene that is relevant for antigen presentation) APC and T cells, are well-established (see, e.g., Murphy, Janeway’s Immunobiology (8thEd.) 2011 Garland Science, NY; chapters 6, 9 and 16). For example, processed antigen peptides originating in the cytosol (e.g., tumor antigen, intracellular pathogen) are generally from about 7 amino acids to about 11 amino acids in length and will associate with class I MHC (HLA) molecules, whereas peptides processed in the vesicular system (e.g., bacterial, viral) will generally vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC (HLA) molecules.

[0276] Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. For example, the term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment. Thus, the term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgGl, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

[0277] The terms " VL" or "VL" and "VH" or "VH" refer to the variable binding region or domain from an antibody light chain and an antibody heavy chain, respectively. In certain embodiments, a VL is a kappa (K) class (also "VK" herein). In certain embodiments, a VL is a lambda (X) class. Like TCR variable domains, the variable domains of antibodies comprise CDRs and framework regions (FRs). There are three CDRs in each antibody variable domain (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRHs and CDRLs, respectively). In certain embodiments, an antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-binding site through their respective CDRs. TCR-mimic antibodies are antibodies (of any isotype e.g., IgG (1, 2, 3, 4), IgE, IgD, IgA, IgM) that are capable of binding to a peptide:MHC complex (z.e., recognizing the peptide:MHC complex and binding thereto). In some embodiments, TCR-mimic antibodies possess antigenspecific, major histocompatibility complex-compatibility or -restriction similar to that of T-cell receptors. TCR-mimic antibodies may be prepared by the hybridoma methodology described by Kohler et al., Nature 256 :495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). TCR-mimic 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), and Noy et al. Expert Rev. Anticancer Ther. 5(3):523-536 (2005); these techniques are incorporated herein in their entireties), for example. TCR-mimic antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004 / 076677A2. Antigenbinding fragments of TCR-mimic antibodies (e.g., a CDR, a VH, a VL, a Fab, a Fd, or the like) are also contemplated. Single-chain fusion proteins and disulfide-engineered proteins comprising an antigen-binding fragment or domain of a TCR-mimic antibody (e.g., scFv, scFab, dsFv, dsFab) are also contemplated.

[0278] In certain embodiments, a binding protein comprises one or more variable domain from an immungolublin superfamily binding protein. In some embodiments, a binding proteim comprises a T cell receptor (TCR) a-chain variable domain (Va) and / or a TCR P-chain variable domain (VP). In some embodiments, a binding proteim comprises one or more variable domain from a TCR-mimic antibody (see e.g. Kurosawa et al., Sci Reports 9:9822 (2019); Trenevska et al. Front. Immunol. (2017) doi.org / 10.3389 / fimmu.2017.01001; Dahan & Reiter, Expert Rec. Mol. Med. 14 e6 (2012) doi.org / 10.1017 / erm.2012.2; Chang et al. Exper Opin Biol Ther 16:929- 982 (2016) doi.org / 10.1080 / 14712598.2016.1176138); Noy et al., Expert Rev. Anticancer Ther. 5(3):523-236 (2005)).

[0279] The term “KRAS-specific binding protein,” as used herein, refers to a protein or polypeptide, such as, for example, a TCR, a scTv, a scTCR, TCR-CAR, dsTv, TCR-mimic antibody, or CAR, that binds to a KRAS peptide antigen or a NRAS peptide antigen or a HRAS peptide antigen (or to a KRAS or NRAS or HRAS peptide antigen:HLA complex, e.g., on a cell surface), and does not bind a peptide that does not contain the KRAS or NRAS or HRAS peptide antigen and does not bind to an HLA complex containing such a peptide.

[0280] Binding proteins of this disclosure, such as TCRs, scTvs, dsTvs, scTCRs, TCR-mimic antibodies and binding fragments thereof, TCR-CARs, and CARs, contain a binding domain specific for a target. A “binding domain” (also referred to as a “binding region” or “binding moiety”), as used herein, refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target (e.g., KRAS or NRAS or HRAS peptide or KRAS or NRAS or HRAS peptide:MHC complex). A binding domain includes any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e. complex comprising two or more biological molecules), or other target of interest. Exemplary binding domains include immunoglobulin variable regions or single chain constructs comprising the same (e.g., single chain TCR (scTCR) or scTv).

[0281] As used herein “specifically binds” or “specific for” refers to an association or union of a binding protein (e.g., TCR receptor) or a binding domain (or fusion protein thereof) to a target molecule with an affinity or Ka(z.e., an equilibrium association constant of a particular binding interaction with units of 1 / M) equal to or greater than 105M'1(which equals the ratio of the on- rate [kon]to the off-rate [koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains (or fusion proteins thereof) may be classified as “high affinity” binding proteins or binding domains (or fusion proteins thereof) or as “low affinity” binding proteins or binding domains (or fusion proteins thereof). “High affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a Kaof at least 107M’1, at least 108M’1, at least 109M’1, at least IO10M’1, at least 1011M’1, at least 1012M’1, or at least 1013M’1. “Low affinity” binding proteins or binding domains refer to those binding proteins or binding domains having a Kaof up to 107M’1, up to 106M’1, up to 105M’1. Alternatively, affinity can be defined as an equilibrium dissociation constant (Ka) of a particular binding interaction with units of M (e.g., 10'5M to IO’13M).

[0282] In certain embodiments, a receptor or binding domain may have “enhanced affinity,” which refers to a selected or engineered receptors or binding domain with stronger binding to a target antigen than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a Ka(equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Ka (dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (koff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof.

[0283] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51 :660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). Binding and binding affinity can also be assessed using, for example, fluorescence intensity observed when a binding protein binds to a labelled HLA-peptide complex or labelled HLA-peptide complex multimer (e.g., tetramer).

[0284] In particular embodiments, a KRAS (or NRAS, or HRAS)-specific binding domain includes a KRAS (or NRAS, or HRAS)-specific scTCR (e.g., single chain a0TCR proteins such as comprising Va-L-VP, VP-L-Va, Va-Ca-L-Va, or Va-L-VP-CP, wherein Va and VP are TCRa and P variable domains respectively, Ca and CP are TCRa and P constant domains, respectively, and L is a linker, such as a linker described herein). In some embodiments, a KRAS (or NRAS, or HRAS)-specific binding domain includes a KRAS (or NRAS, or HRAS)-specific scTv (e.g., single chain TCR variable domains proteins such as Va-L-VP or VP- L-Va wherein Va and VP are TCRa and P variable domains respectively, and L is a linker, such as a linker described herein).

[0285] The term “functional avidity”, as used herein, refers to a biological measure or activation threshold of an in vitro immune cell (e.g., T cell, NK cell, NK-T cell) response to a given concentration of a ligand, wherein the biological measures can include cytokine production (e.g, IFN-y production, IL-2 production, etc.), cytotoxic activity, activation markers (e.g, CD137, Nur77) and proliferation. For example, T cells that biologically (immunologically) respond in vitro to a low antigen dose by, for example, producing cytokines, exhibiting cytotoxic activity, or proliferating are considered to have high functional avidity, while T cells having lower functional avidity require higher amounts of antigen before an immune response, similar to the high-avidity T cells, is elicited. It will be understood that functional avidity is different from affinity and avidity. Affinity refers to the strength of any given bond between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to multiple antigens - in this case, the strength of the overall connection is the avidity.

[0286] Numerous correlations exist between the functional avidity and the effectiveness of an immune response. Some ex vivo studies have shown that distinct T cell functions (e.g., proliferation, cytokines production, etc.) can be triggered at different thresholds (see, e.g., Betts et al., J. Immunol. 172:6407, 2004; Langenkamp et al., Eur. J. Immunol. 32:2046, 2002). Factors that affect functional avidity can include (a) the affinity of a TCR for the pMHC- complex, that is, the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167:2577, 2001), (b) expression levels of the TCR, and, in some embodiments, CD4 or CD8 co receptors, on the host cell and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273: 104, 1996), as well as expression levels of molecules that attenuate T cell function and TCR signaling.

[0287] The concentration of antigen needed to induce a half-maximum response (e.g., production of a cytokine or activation marker by a host cell; fluorescence intensity when binding to a labeled peptide:HLA multimer) between the baseline and maximum response after a specified exposure time is referred to as the “half maximal effective concentration” or “EC50”. The EC50 value is generally presented as a molar (moles / liter) amount, but it is often converted into a logarithmic value as follows - logio(EC50). For example, if the EC50 equals 1 pM ( 1 O’6M), the logio(EC50) value is -6. Another value used is pEC50, which is defined as the negative logarithm of the EC50 (-logio(EC50)). In the above example, the EC50 equaling 1 pM has a pEC50 value of 6. In certain embodiments, the functional avidity of a binding protein of this disclosure will comprise a measure of an ability of the binding protein to promote activation and / or IFNy production by T cells, which can be measured using assays known in the art and described herein. In certain embodiments, functional avidity will comprise a measure of the ability of the binding protein, upon binding to antigen, to activate a host cell, such as a T cell.

[0288] Binding proteins disclosed herein can comprise high functional avidity that can, for example, facilitate elicitation of immune cell effector functions (e.g., activation, proliferation, cytokine production, and / or cytotoxicity) against even lower levels of a KRAS G12V peptide the KRAS G12V mutant peptide of SEQ ID NO: 2, when presented in an HLA-A*02:01.

[0289] Also contemplated are fusion proteins comprising a scTCR or scTv or dsTv of the present disclosure linked to a constant domain (e.g. heavy chain constant domain or combination thereof, such as a Fc, a CH2, a CH3, a CH4, and / or a CHI) of an antibody (e.g., IgG (1, 2, 3, 4), IgE, IgD, IgA, IgM, and variants thereof) or a fragment thereof (e.g., a fragment that, in some embodiments, retains binding to one or more Fc receptors, to Clq, to Protein A, to Protein G, or any combination thereof), and including immunoglobulin heavy chain monomers and multimers, such as Fc dimers; see, e.g., Wong et al., J. Immunol. 198: 1 Supp. (2017). Variant Fc polypeptides comprising mutations that enhance, reduce, or abrogate binding to or by, e.g., FcRn or other Fc receptors, are known and are contemplated within this disclosure.

[0290] In certain embodiments, a binding protein or fusion protein (e.g., TCR, scTCR, CAR) of the present disclosure is expressed by a host cell (e.g., by a T cell, NK cell, or NK-T cell heterologously expressing the binding protein or fusion protein). Avidity of such a host cell for a KRAS (or NRAS, or HRAS) peptide antigen or KRAS (or NRAS, or HRAS) peptide antigen:HLA complex can be determined by, for example, exposing the host cell to the peptide, or to a peptide:HLA complex (e.g., organized as a tetramer), or to an antigen-presenting cell (APC) that presents the peptide to the host cell, optionally in a peptide:HLA complex, and then measuring an activity of the host cell, such as, for example, production or secretion of cytokines (e.g., IFN-y; TNFa); increased expression of host cell signaling or activation components (e.g., CD137 (4-1BB)); proliferation of the host cell; or killing of the APC (e.g., using a labeled- chromium release assay).

[0291] As used herein, “nucleic acid” or “nucleic acid molecule” or “polynucleotide” refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, polynucleotides, fragments thereof generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and also to fragments generated by any of ligation, scission, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphonothioate, phosphonodithioate, phosphonoselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid monomers may comprise phosphorothioate linkages, phosphorodithioate linkages, or phosphoroselenoate linkages, or any combination thereof. Nucleic acid molecules can be either single-stranded or double-stranded.

[0292] Nucleic acid molecules include polyribonucleic acid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double-stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense strand).

[0293] A nucleic acid molecule encoding an amino acid sequence can comprise any nucleotide sequence that encodes the amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post- transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.

[0294] In some embodiments, a polynucleotide or expression construct of the present disclosure comprises DNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises RNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises mRNA. In some embodiments, a polynucleotide or expression construct of the present disclosure comprises circRNA, saRNA, taRNA, or any combination thereof.

[0295] The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system (or host animal), is isolated. Such a nucleic acid could be part of a vector and / or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. In some embodiments, an isolated binding protein, polynucleotide, vector, or host cell is provided.

[0296] The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (“leader and trailer”) as well as intervening sequences (introns) between individual coding segments (exons).

[0297] As used herein, the terms “recombinant”, “engineered”, and “modified” refer to a cell, microorganism, nucleic acid molecule, polypeptide, protein, plasmid, or vector that has been modified by introduction of an exogenous nucleic acid molecule, or refers to a cell or protein or microorganism that has been genetically engineered by human intervention — that is, modified by introduction of a heterologous nucleic acid molecule or introduction of a non-native linkage, coupling, amino acid, conjugation site, glycan, chemical modification, or the like, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering. Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material. Exemplary modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.

[0298] As used herein, “mutation” refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s). In certain embodiments, a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof. A mutation can result in a “variant” of a protein.

[0299] A “conservative substitution” is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97 / 09433 at page 10; Lehninger, Biochemistry, 2ndEdition; Worth Publishers, Inc. NY, NY, pp.71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0300] In certain embodiments, proteins (e.g., binding protein, immunogenic peptide) according to the present disclosure comprise a variant sequence as compared to a reference sequence (e.g., a variant TCR CDR (e.g., CDR3P) as compared to a reference TCR CDR (e.g., CDR3P) disclosed herein). As used herein, a “variant” amino acid sequence, peptide, or polypeptide, can refer to an amino acid sequence (or peptide or polypeptide) having one, two, or three amino acid substitutions, deletions, and / or insertions as compared to a reference amino acid sequence. In certain embodiments, a variant amino acid sequence, peptide, or polypeptide, retains substantially a same functionality (e.g., binding specificity and affinity for a peptide:HLA complex) as the reference molecule; for example, a variant TCR fragment as disclosed herein retains about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or 100% of the antigen-binding specificity and affinity as compared to a reference TCR binding fragment.

[0301] An “altered domain” or “altered protein” refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRa chain, TCRP chain, TCRa constant domain, TCRP constant domain) of at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%).

[0302] Altered domains or altered proteins or derivatives can include those based on all possible codon choices for the same amino acid and codon choices based on conservative amino acid substitutions. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (ala; A), serine (ser; S), threonine (thr; T); 2) aspartic acid (asp; D), glutamic acid (glu; E); 3) asparagine (asn; N), glutamine (gin; Q); 4) arginine (arg; R), lysine (lys; K); 5) Isoleucine (ile; I), leucine (L), methionine (met; M), valine (val; V); and 6) phenylalanine (phe; F), tyrosine (tyr; Y), tryptophan (trp; W). (See also WO97 / 09433 at page 10, Lehninger, Biochemistry, 2ndEdition, Worth Publishers, Inc., NY, NY, pp. 71-77, 1975; Lewin Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p.8, 1990; Creighton, Proteins, W.H. Freeman and Company 1984). In addition, individual substitutions, deletions, or additions that alter, add or delete, a single amino acid or a small percentage of amino acids in an encoded sequence are also “conservative substitutions.”

[0303] The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule. A “transgene” or “transgene construct” refers to a construct that contains two or more genes operably linked in an arrangement that is not found in nature. The term “operably- linked” (or “operably linked” herein) refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence when it can affect the expression of that coding sequence (z.e., the coding sequence is under the transcriptional control of the promoter). “Unlinked” means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other. In some embodiments, the genes present in a transgene are operably linked to an expression control sequence (e.g., a promoter).

[0304] A construct (e.g., a transgene) can be present in a vector (e.g., a bacterial vector, a viral vector) or can be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors can be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that can include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. Examples of vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acid molecules to which they are linked (expression vectors). Vectors useful in the compositions and methods of this disclosure are described further herein. The term “expression”, as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof.

[0305] The term “introduced” in the context of inserting a nucleic acid molecule into a cell, means “transfection”, or “transformation”, or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule can be incorporated into the genome of a cell (e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). "Introduced" includes delivery of a polynucleotide using a lipid nanoparticle or other lipid-based delivery vehicle, electroporation, circular RNA, or the like.

[0306] As used herein, “heterologous” or “exogenous” nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but can be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule is added (z.e., not endogenous, or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can be present in multiple copies. In addition, “heterologous” refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity. Moreover, a cell comprising a “modification” or a “heterologous” polynucleotide or binding protein includes progeny of that cell, regardless of whether the progeny were themselves transduced, transfected, or otherwise manipulated or changed.

[0307] As described herein, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, as disclosed herein, a host cell can be modified to express one or more heterologous or exogenous nucleic acid molecule encoding desired TCR specific for a KRAS G12V 5-14 peptide (e.g., TCRa and TCR0) and optionally, as disclosed herein, also encoding a CD8 co-receptor polypeptide comprising a a chain, a P chain, or a portion thereof, such as an extracellular portion capable of binding to MHC. A heterologous polynucleotide encode two or more polypeptides, each two successive polypeptides being separated by, for example, an amino acid sequence comprising a selfcleaving peptide (in any of the presently disclosed embodiments, a self-cleaving peptide can comprise a linker N-terminal and / or C-terminal to the self-cleaving (e.g., viral 2A, such as P2A, E2A, F2A, or T2A, or an engineered variant thereof) sequence. Non-limiting examples of linkers include GSG, GPP, PGP, and AAA), a protease (e.g., furin) cleavage site, or both, or any combination thereof. When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, and not necessarily to the number of separate nucleic acid molecules introduced into a host cell.

[0308] As used herein, the term “endogenous” or “native” refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated, or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell. For example, an endogenous control sequence from a first gene (e.g., a promoter, translational attenuation sequences) can be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.

[0309] The term “homologous” or “homolog” refers to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene, and can optionally have an altered expression level, a different sequence, an altered activity, or any combination thereof.

[0310] “Sequence identity,” or “percent sequence identity,” as used herein, refers to the percentage of amino acid residues or nucleobases in one sequence that are identical with the amino acid residues or nucleobases (respectively) in a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The percentage sequence identity values can be generated using the NCBI BLAST 2.0 software as defined by Altschul et al. (1997), NucL Acids Res. 25:3389-3402, with the parameters set to default values. Additionally or alternatively, the degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs designed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, Needle (EMBOSS), Stretcher (EMBOSS), GGEARCH2SEQ, Water (EMBOSS), Matcher (EMBOSS), LALIGN, SSEARCH2SEQ, or another suitable method or algorithm. A global alignment algorithm, such as a Needleman and Wunsch algorithm, can be used to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Default settings can be used.

[0311] To generate similarity scores for two amino acid sequences, scoring matrices can be used that assign positive scores for some non-identical amino acids (e.g., conservative amino acid substitutions, amino acids with similar physio-chemical properties, and / or amino acids that exhibit frequent substitutions in orthologs, homologs, or paralogs), Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0312] Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and are preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. In some embodiments, variant nucleic acid molecules are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and are preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a binding protein or a binding domain thereof having a functionality described herein, such as binding a target molecule. In some contexts, the term “variant” as used herein, refers to at least one fragment of the full-length sequence referred to, more specifically one or more amino acid or nucleic acid sequence which is, relative to the full-length sequence, truncated at one or both termini by one or more amino acids. Such a fragment includes or encodes a peptide having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, or 200 successive amino acids of the original sequence or a variant thereof. The total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids.

[0313] In some embodiments, the term “variant” relates not only to at least one fragment, but also to a polypeptide or a fragment thereof including amino acid sequences that are at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the reference amino acid sequence referred to or the fragment thereof, wherein amino acids other than those essential for the biological activity or the fold or structure of the polypeptide are deleted or substituted, one or more such essential amino acids are replaced in a conservative manner, and / or amino acids are added such that the biological activity of the polypeptide is preserved. The state of the art includes various methods that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity (see, e.g., Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition). In some embodiments, the Clustal W software can be used using default settings (Larkin, M. A., et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948).

[0314] In certain embodiments, variants may, in addition, include chemical modifications, for example, isotopic labels or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation and the like. Methods for modifying polypeptides are known and in general will be employed so as not to abolish or substantially diminish a desired activity of the polypeptide.

[0315] In an embodiment, the term “variant” of a nucleic acid molecule includes nucleic acids the complementary strand of which hybridizes, for example, under stringent conditions, to the reference or wild type nucleic acid. Stringency of hybridization reactions is readily determinable by one of ordinary skill in the art, and in general is an empirical calculation dependent on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes less so. Hybridization generally depends on the ability of denatured DNA to reanneal to complementary strands present in an environment below their melting temperature: the higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which may be used. As a result, higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperature less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel, F. M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Moreover, the person skilled in the art may follow the instructions given in the manual Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, K. H. (1991) International Journal of Systematic Bacteriology 41 : 255-260 on how to identify DNA sequences by means of hybridization. In an embodiment, stringent conditions are applied for any hybridization, i.e., hybridization occurs only if the probe is 70% or more identical to the target sequence. Probes having a lower degree of identity with respect to the target sequence may hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example, lowering the concentration of salt to 2* SSC or, optionally and subsequently, to 0.5* SSC, while the temperature is, for example, about 50 °C-68 °C, about 52 °C-68 °C, about 54 °C-68 °C, about 56 °C-68 °C, about 58 °C-68 °C, about 60 °C-68 °C, about 62 °C-68 °C, about 64 °C-68 °C, or about 66 °C-68 °C. In an embodiment, the temperature is about 64 °C-68 °C or about 66 °C-68 °C. It is possible to adjust the concentration of salt to 0.2* SSC or even O.lx SSC. Nucleic acid sequences having a degree of identity with respect to the reference or wild type sequence of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% may be isolated. In an embodiment, the term variant of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that encodes the same amino acid sequence and variants thereof as the reference nucleic acid sequence, in line with, e.g., the degeneracy of the genetic code.

[0316] A “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs, in some contexts slightly, in composition (e.g., one base, atom or functional group is different, added, or removed; or one or more amino acids are mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% level of activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has “similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant), avidity, or activation of a host cell. TCR signaling or an activity stimulated thereby (e.g. as exemplified herein, such as measuring IFN-g production, IL-2 production, intracellular calcium flux, cellular avidity as determined by the percentage of cells in a sample that bind to antigen, proliferation, specific cytotoxicity against a target cell, NF AT expression, NFkB expression, AP-1 expression, Nur77 expression).

[0317] As used herein, a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, motif, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, or at least 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).

[0318] A “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity), such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release). Functional variants of specifically disclosed binding proteins and polynucleotides are contemplated.

[0319] An “altered domain” or “altered protein” refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRa chain, TCRP chain, TCRa constant domain, or TCRP constant domain) of at least 85% (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%). In one aspect, the present disclosure provides a binding protein, comprising a T cell receptor (TCR) a chain variable (Va) domain and a TCR P chain variable (VP) domain, wherein the binding protein is capable of binding to a KRAS G12V peptide antigen: HL A- A2 complex, wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2. In some embodiments, the petpdie comprises one, two, or three methyl groups at a lysine corresponding to position 5 of SEQ ID NO: 1. In certain embodiments, the HLA comprises an HLA-A*2:01. In any of the presently disclosed embodiments, the binding protein can be heterologously expressed by a human immune system cell, such as, for example, a T cell.

[0320] In certain embodiments, the Va domain and / or the VP domain are each independently human, humanized, or chimeric, and are preferably each human. In some embodiments, the Va domain is human and the VP domain is human. Binding proteins, compositions, and methods disclosed herein can utilize a Va domain, VP domain, or CDRs therefrom derived from a human subject, for example, from sequencing of an isolated T cell or population thereof from a human subject. TCR Va domains, VP domains, and CDRs therefrom isolated from a human subject can have advantageous properties over variable domains and CDRs from other sources, such as mice transgenic for a single human HLA allele. For example, Va domains, VP domains, and CDRs derived from a human subject can have undergone negative thymic selection against substantially the whole human peptidome presented by a full set of human HLA molecules in vivo, which can reduce the likelihood that the binding protein is cross-reactive to other human self-antigens.

[0321] In some embodiments, a binding protein comprises one or more variable domains or one or more CDRs derived from (e.g., identified in) a T cell of a subject (e.g., a human subject) having a disease, such as a cancer. In some embodiments, a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a human subject having a cancer disclosed herein. In some embodiments, a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a subject (e.g., a human subject) having a disease associated with a KRAS G12V mutation. In some embodiments, a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a subject (e.g., a human subject) with a cell that comprises a KRAS G12V mutation and a HLA-A2.

[0322] In some embodiments, a binding protein comprises one or more variable domains or one or more CDRs derived from a T cell of a healthy subject (e.g., a healthy human subject). In some embodiments, a healthy subject lacks a specific pathological diagnosis (e.g., disease diagnosis, such as a cancer diagnosis). In some embodiments, a healthy subject lacks a specific pathological diagnosis, but comprises a different pathological diagnosis, for example, lacks a cancer diagnosis but comprises a diagnosis of hypertension or type II diabetes.

[0323] Presently disclosed binding proteins are capable of being heterologously expressed by host cells, such as, for example, human immune cells, such as T cells. Furthermore, expression of a presently disclosed binding protein can confer advantageous properties upon a host cell; e.g., having binding specificity for a KRAS G12V peptide antigen:HLA-A2 complex of the present disclosure, improved activation, proliferation, or killing activity in the presence of a KRAS G12V peptide antigen:HLA-A2 presenting tumor cell, or the like.

[0324] For example, in certain embodiments, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell is capable of specifically killing a HLA-A2+tumor cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2. Killing of a target cell can be determined, for example, the Incucyte® bioimaging platform (Essen Bioscience). In certain embodiments, this platform uses activated caspase and labelled (e.g., RapidRed or NucRed) tumor cell signals, wherein overlap is measured and increased overlap area equals tumor cell death by apoptosis. Killing can also be determined using a 4-hour assay in which target cells are loaded with labeled chromium (51Cr), and51Cr in the supernatant is measured, e.g., following 4-hour co-incubation with an immune cell expressing a binding protein of the present disclosure. In certain embodiments, a killing assay can be performed using an effectortarget cell ratio of 0.5: 1, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 20: 1, 25: 1, 50: 1, or 100: 1, or the like.

[0325] In any of the presently disclosed embodiments, when the binding protein is expressed by an immune cell (e.g, a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell has elevated expression of Nur77 when in the presence of a HLA- A2+tumor cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO:2, optionally in the further presence of exogenous IFN-y, wherein the Nur77 expression is elevated as compared to: (i) Nur77 expression by a reference immune cell (z.e., of the same cell type as, and otherwise phenotypically and / or genotypically at least substantially identical or functionally equivalent to, the immune cell expressing the binding protein) not expressing the binding protein, when the reference immune cell is in the presence of the tumor cell; and / or (ii) Nur77 expression by the immune cell expressing the binding protein when not in the presence of the tumor cell and / or when not in the presence of an antigen-presenting cell expressing a KRAS G12V antigen peptide:HLA-A2 complex, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2, and wherein the HLA-A2 is optionally HLA-A*02:01. Expression of Nur77 can be determined, for example, using a transgenic expression construct comprising a Nur77 locus operably linked to a sequence encoding a reporter construct; e.g., dTomato (see Ahsouri and Weiss, J Immunol 79S(2):657-668 (2017)).

[0326] In any of the presently disclosed embodiments, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the immune cell has elevated expression of CD137 (also known as 4-1BB) when in the presence of a HLA-A*02+tumor cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2, optionally in the further presence of exogenous IFN-y, wherein the CD137 expression is elevated as compared to: (i) CD137 expression by a reference immune cell not expressing the binding protein, when the reference immune cell is in the presence of the tumor cell; and / or (ii) CD 137 expression by the immune cell expressing the binding protein when not in the presence of the tumor cell and / or when not in the presence of an antigen-presenting cell expressing a KRAS G12V peptide antigen:HLA-A2 complex, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2, and wherein the HLA-A2 is optionally HLA-A*0-2:01. CD137 expression can be determined using, for example, flow cytometry using a labeled anti- CD137 antibody. In certain embodiments, CD137 is measured following a 16-hour assay in which the immune cell is co-incubated with or stimulated with peptide or a target cell expressing the peptide.

[0327] In any of the presently disclosed embodiments: (i) the binding protein is encoded by a polynucleotide that is heterologous to the immune cell; (ii) the immune cell comprises a human CD8+T cell, a human CD4+ T cell, or both; (iii) the tumor cell expressing a peptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2 is HLA-A2+; and / or (iv) the tumor cell comprises a DAN-G (pancreatic adenocarcinoma) or CFPAC1 (pancreatic adenocarcinoma) cell.

[0328] In certain embodiments, the binding protein is capable of binding to the KRAS G12V peptide antigen:HLA-A2 complex independent of, or in the absence of, CD8. CD8-independent binding can be determined by expressing the binding protein in a CD8-negative cell (e.g., a CD4+T cell, a Jurkat cell, or the like) and identifying binding of the cell to a target. In some embodiments, a binding protein is provided that comprises: (a) a T cell receptor (TCR) a chain variable (Va) domain comprising the complementarity determining region 3 (CDR3a) amino acid sequence set forth in any one of SEQ ID NOs: 37, 53, and 69, or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions; and / or (b) a TCR P chain variable (VP) domain comprising the CDR3P amino acid sequence set forth in any one of SEQ ID NOs: 45, 61, and 77, respectively (i.e. Va SEQ ID NO: 37 with VP SEQ ID NO: 45), or a variant thereof having one, two, or three, optionally conservative, amino acid substitutions, wherein the binding protein is capable of binding to a KRAS G12V peptide antigen:HLA-A2 complex, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequenceof SEQ ID NO: 2), and wherein the HLA-A2 optionally comprises an HLA- A*02:01. The binding protein can comprise the Va domain and the VP domain.

[0329] The Va domain and / or the VP domain can be human, humanized, or chimeric, and is preferably human.

[0330] In certain embodiments, the binding protein comprises the CDR3a and CDR3P amino acid sequences set forth in SEQ ID NOs.: (i) 12and 32, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (ii) 14 and 232, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iii) 16 and 32, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (iv) 18 and 32, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (v) 20 and 32, respectively, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vi) 22 and 32, respectively, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (vii) 24 and 32, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (viii) 40 and 48, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; (ix) 56 and 64, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions; or (x) 72 and 80, respectively, or variants thereof having one, two, or three, optionally conservative, amino acid substitutions. In some embodiments, the CDR3a and the CDR3P cannot both have the amino acid sequences set forth in SEQ ID NOs: 12 and 32, respectively. In certain embodiments, the binding protein comprises the CDR3a and CDR3P amino acid sequences set forth in SEQ ID NOs.: (i) 14 and 32, respectively; (ii) 16 and 32, respectively; (iii) 18 and 32, respectively; (iv) 20 and 32, respectively; (v) 22 and 32, respectively; (vi) 24 and 32, respectively; (viii) 40 and 48, respectively; (ix) 56 and 64, respectively; or (x) 72 and 80, respectively.

[0331] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 11, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO:24, or a variant thereof having one or two, optionally conservative, amino acid substitutions; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 25, or a variant thereof having one or two, optionally conservative, amino acid substitutions; or (v) any combination of (i)-(iv). The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0332] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 10; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 11; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 30; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 31; or (v) any combination of (i)-(iv).

[0333] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 10; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 11; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 30; and (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 31.

[0334] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 38; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 39; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 46; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 47; or (v) any combination of (i)-(iv).

[0335] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 38; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 39; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 46; and (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 47. In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 54; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 55; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 62; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 63; or (v) any combination of (i)-(iv).

[0336] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 54; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 55; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 62; and (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 63.

[0337] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 70; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 71; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 78; (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 79; or (v) any combination of (i)-(iv).

[0338] In some embodiments, the binding protein further comprises: (i) in the Va domain, the CDRla amino acid sequence set forth in SEQ ID NO: 70; (ii) in the Va domain, the CDR2a amino acid sequence set forth in SEQ ID NO: 71; (iii) in the VP domain, the CDRip acid sequence set forth in SEQ ID NO: 78; and (iv) in the VP domain, the CDR2P acid sequence set forth in SEQ ID NO: 79.

[0339] In certain embodiments, the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 10; 11; 14, 16, 18, 20, 22, or 24; 30, 31, and 32, respectively. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0340] In other embodiments, the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 38, 39, 40, 46, 47, and 48, respectively.

[0341] In other embodiments, the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 54, 55, 56, 62,63, and 64, respectively. In other embodiments, the binding protein comprises the CDRla, CDR2a, CDR3a, CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.: 70, 71, 72, 78 79, and 80, respectively.

[0342] In some embodiments, a binding protein disclosed herein comprises a CDRla, CDR2a, CDR3a, CDRip, CDR2P, and / or CDR3P as identified by the Kabat method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 45; SEQ ID NO: 53 and SEQ ID NO: 61; or SEQ ID NO: 69 and SEQ ID NO: 77. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9. .

[0343] In some embodiments, a binding protein disclosed herein comprises a CDRla, CDR2a, CDR3a, CDRip, CDR2P, and / or CDR3P as identified by the Chothia method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 45; SEQ ID NO: 53 and SEQ ID NO: 61; or SEQ ID NO: 69 and SEQ ID NO: 77. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0344] In some embodiments, a binding protein disclosed herein comprises a CDRla, CDR2a, CDR3a, CDRip, CDR2P, and / or CDR3P as identified by the EU method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 45; SEQ ID NO: 53 and SEQ ID NO: 61; or SEQ ID NO: 69 and SEQ ID NO: 77. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0345] In some embodiments, a binding protein disclosed herein comprises a CDRla, CDR2a, CDR3a, CDRip, CDR2P, and / or CDR3P as identified by the IMGT method or numbering scheme (including IMGT and / or IMGT-junction for CDR3) from the variable domain sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 45; SEQ ID NO: 53 and SEQ ID NO: 61; or SEQ ID NO: 69 and SEQ ID NO: 77. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, a binding protein disclosed herein comprises a CDRla, CDR2a, CDR3a, CDRip, CDR2P, and / or CDR3P as identified by the Enhanced Chothia method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 45; SEQ ID NO: 53 and SEQ ID NO: 61; or SEQ ID NO: 69 and SEQ ID NO: 77. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0346] In some embodiments, a binding protein disclosed herein comprises a CDRla, CDR2a, CDR3a, CDRip, CDR2P, and / or CDR3P as identified by the Aho method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, 15, 17, 19, 21, or 23 and SEQ ID NO: 32; SEQ ID NO: 37 and SEQ ID NO: 45; SEQ ID NO: 53 and SEQ ID NO: 61; or SEQ ID NO: 69 and SEQ ID NO: 77. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0347] In some embodiments, the binding protein comprises a CDRla that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 10, 38, 54, or

[0348] 70, or a CDRla sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69. In some embodiments, the binding protein comprises a CDR2a that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 11, 39, 55, or

[0349] 71, or a CDR2a sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69. In some embodiments, the binding protein comprises a CDR3a that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 14, 16, 18, 20, 22, 24, 40, 56, or 72, or a CDR3a sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the binding protein comprises a CDRip that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 30, 46, 62, or 78, or a CDRip sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. In some embodiments, the binding protein comprises a CDR2P that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 31, 47, 63, or 79, or a CDR2P sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. In some embodiments, the binding protein comprises a CDR3P that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 32, 48, 64, or 80, or a CDR3P sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78.

[0350] In some embodiments, the binding protein comprises a CDRla that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 10, 38, 54, or 70, or a CDRla sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69. The substitution(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0351] In some embodiments, the binding protein comprises a CDR2a that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 11, 39, 55, or 71, or a CDR2a sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69. The substitution(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0352] In some embodiments, the binding protein comprises a CDR3a that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 14, 16, 18, 20, 22, or 24, or a CDR3a sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69 or 39. The substitution(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative. The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0353] In some embodiments, the binding protein comprises a CDRip that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 30, 46, 62, or 78, or a CDRip sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. The substitution(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0354] In some embodiments, the binding protein comprises a CDR2P that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 31, 47, 63, or 79, or a CDR2P sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. The substitution(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0355] In some embodiments, the binding protein comprises a CDR3P that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 32, 48, 64, or 80 or a CDR3P sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. The substitution(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0356] In some embodiments, the binding protein comprises a CDRla that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 10, 38, 54, or 70, or a CDRla sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus of the CDR, the C- terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0357] In some embodiments, the binding protein comprises a CDR2a that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 11, 39, 55, or 71, or a CDR2a sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus of the CDR, the C- terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0358] In some embodiments, the binding protein comprises a CDR3a that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 14, 16, 18, 20, 22, or 24, or a CDR3a sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23 37, 53, or 69 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0359] In some embodiments, the binding protein comprises a CDRip that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 30, 46, 62, or 78, or a CDRip sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. The insertion(s) and / or deletion(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0360] In some embodiments, the binding protein comprises a CDR2P that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs:31, 47, 63, or 79, or a CDR2P sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. The insertion(s) and / or deletion(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. In some embodiments, the binding protein comprises a CDR3P that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 32, 48, 64, or 80or a CDR3P sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 62, or 78. The insertion(s) and / or deletion(s) can be at the N-terminus of the CDR, the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0361] A binding protein disclosed herein can comprise one or more framework regions (FRs). For example, a binding protein can comprise a variable domain comprising three CDRs and four FRs, or two variable domains each comprising three CDRs and four FRs. Illustrative FR amino acid sequences are provided by SEQ ID NOs: 25-28, 33-36, 41-44, 49-52, 57-60, 65-68, 73-76, and 81-84. A framework region used in a binding protein can be a mammalian framework region. A framework region used in a binding protein can be a human framework region. A framework region used in a binding protein can be an engineered framework region.

[0362] A binding protein can comprise an FR1, an FR2, and FR3, and / or an FR4 disclosed herein. In some embodiments, a binding protein comprises a Va comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 25, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 26, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 27 or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 28, or a variant thereof. In some embodiments, the binding protein comprises a Va comprising the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15, 17, 19, 21, or 23. In some embodiments, the Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12, or the Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0363] In some embodiments, a binding protein comprises a VP comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 33, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 34, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO:35, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 36, or a variant thereof. In some embodiments, the binding protein comprises a VP comprising the amino acid sequence set forth in SEQ ID NO: 29.

[0364] A binding protein can comprise an FR1, an FR2, and FR3, and / or an FR4 disclosed herein. In some embodiments, a binding protein comprises a Va comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 41, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 42, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 43, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 44, or a variant thereof. In some embodiments, the binding protein comprises a Va comprising the amino acid sequence set forth SEQ ID NO: 37.

[0365] In some embodiments, a binding protein comprises a VP comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 49, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 50, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 51, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 52, or a variant thereof. In some embodiments, the binding protein comprises a VP comprising the amino acid sequence set forth in SEQ ID NO: 45.

[0366] A binding protein can comprise an FR1, an FR2, and FR3, and / or an FR4 disclosed herein. In some embodiments, a binding protein comprises a Va comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 57, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 58, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 59, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 60, or a variant thereof. In some embodiments, the binding protein comprises a Va comprising the amino acid sequence set forth SEQ ID NO: 53.

[0367] In some embodiments, a binding protein comprises a VP comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 65, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 66, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 67, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 68, or a variant thereof. In some embodiments, the binding protein comprises a VP comprising the amino acid sequence set forth in SEQ ID NO: 61.

[0368] A binding protein can comprise an FR1, an FR2, and FR3, and / or an FR4 disclosed herein. In some embodiments, a binding protein comprises a Va comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 73, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 74, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 75, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 76, or a variant thereof. In some embodiments, the binding protein comprises a Va comprising the amino acid sequence set forth SEQ ID NO: 69.

[0369] In some embodiments, a binding protein comprises a VP comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 81, or a variant thereof, an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 82, or a variant thereof, an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 83, or a variant thereof, and an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO: 84, or a variant thereof. In some embodiments, the binding protein comprises a VP comprising the amino acid sequence set forth in SEQ ID NO: 77.

[0370] In some embodiments, a binding protein comprises a Va domain comprising an FR1, FR2, FR3, and FR4 as identified by the Kabat method or numbering scheme from the variable domain sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. In some embodiments, a binding protein comprises a VP domain comprising an FR1, FR2, FR3, and FR4 as identified by the Kabat method from the variable domain sequence of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0371] In some embodiments, a binding protein comprises a Va domain comprising an FR1, FR2, FR3, and FR4 as identified by the Chothia method or numbering scheme from the variable domain sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. In some embodiments, a binding protein comprises a VP domain comprising an FR1, FR2, FR3, and FR4 as identified by the Chothia method from the variable domain sequence of any one of SEQ ID NOs: 29, 45, 61, or 77. In some embodiments, a binding protein comprises a Va domain comprising an FR1, FR2, FR3, and FR4 as identified by the EU method or numbering scheme from the variable domain sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. In some embodiments, a binding protein comprises a VP domain comprising an FR1, FR2, FR3, and FR4 as identified by the EU method from the variable domain sequence of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0372] In some embodiments, a binding protein comprises a Va domain comprising an FR1, FR2, FR3, and FR4 as identified by the IMGT method or numbering scheme from the variable domain sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. In some embodiments, a binding protein comprises a VP domain comprising an FR1, FR2, FR3, and FR4 as identified by the IMGT method from the variable domain sequence of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0373] In some embodiments, a binding protein comprises a Va domain comprising an FR1, FR2, FR3, and FR4 as identified by the Enhanced Chothia method or numbering scheme from the variable domain sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. In some embodiments, a binding protein comprises a VP domain comprising an FR1, FR2, FR3, and FR4 as identified by the Enhanced Chothia method from the variable domain sequence of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0374] In some embodiments, a binding protein comprises a Va domain comprising an FR1, FR2, FR3, and FR4 as identified by the Aho method or numbering scheme from the variable domain sequence of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. In some embodiments, a binding protein comprises a VP domain comprising an FR1, FR2, FR3, and FR4 as identified by the Aho method from the variable domain sequence of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0375] In some embodiments, the binding protein comprises a Va domain comprising an FR1 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 25, 41, 57, or 73, or an FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69.

[0376] In some embodiments, the binding protein comprises a Va domain comprising an FR2 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 26,42,58, or 74, or an FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69.

[0377] In some embodiments, the binding protein comprises a Va domain comprising an FR3 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 27, 43, 59, or 75, or an FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69.

[0378] In some embodiments, the binding protein comprises a Va domain comprising an FR4 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 28, 44, 60, or 76, or an FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69.

[0379] In some embodiments, the binding protein comprises a VP domain comprising an FR1 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 33, 49, 65, or 81, or an FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0380] In some embodiments, the binding protein comprises a VP domain comprising an FR2 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 34, 50, 66, or 82, or an FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0381] In some embodiments, the binding protein comprises a VP domain comprising an FR3 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 35, 51, 67, or 83, or an FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77.

[0382] In some embodiments, the binding protein comprises a VP domain comprising an FR4 that comprises, consists essentially of, or consists of the amino acid sequence of any one of SEQ ID NOs: 36, 52, 68, or 84, or an FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. In some embodiments, the binding protein comprises a Va domain comprising an FR1 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 925, 41,

[0383] 57, or 73, or an FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, nonconservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0384] In some embodiments, the binding protein comprises a Va domain comprising an FR2 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 926, 42,

[0385] 58, or 74, or an FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, nonconservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0386] In some embodiments, the binding protein comprises a Va domain comprising an FR3 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 27, 43,

[0387] 59, or 75, or an FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, nonconservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0388] In some embodiments, the binding protein comprises a Va domain comprising an FR4 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 28, 44,

[0389] 60, or 76, or an FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, nonconservative, or a combination thereof. In some embodiments, the substitutions are conservative. In some embodiments, the binding protein comprises a VP domain comprising an FR1 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs:33, 49,

[0390] 65, or 81, or an FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0391] In some embodiments, the binding protein comprises a VP domain comprising an FR2 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 34, 50,

[0392] 66, or 82, or an FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0393] In some embodiments, the binding protein comprises a VP domain comprising an FR3 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 35, 51,

[0394] 67, or 83, or an FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0395] In some embodiments, the binding protein comprises a VP domain comprising an FR4 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of any one of SEQ ID NOs: 36, 52,

[0396] 68, or 84, or an FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The substitution(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative. In some embodiments, the binding protein comprises a Va domain comprising an FR1 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 25, 41, 57, or 73, or an FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The insertion(s) and / or deletion(s) can be at the N-terminus, the C- terminus, within the amino acid sequence, or a combination thereof.

[0397] In some embodiments, the binding protein comprises a Va domain comprising an FR2 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 26, 42, 58, or 744, or an FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The insertion(s) and / or deletion(s) can be at the N-terminus, the C- terminus, within the amino acid sequence, or a combination thereof.

[0398] In some embodiments, the binding protein comprises a Va domain comprising an FR3 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 27, 43, 59, or 75, or an FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The insertion(s) and / or deletion(s) can be at the N-terminus, the C- terminus, within the amino acid sequence, or a combination thereof.

[0399] In some embodiments, the binding protein comprises a Va domain comprising an FR4 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 28, 44, 60 or 76, or an FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The insertion(s) and / or deletion(s) can be at the N-terminus, the C- terminus, within the amino acid sequence, or a combination thereof.

[0400] In some embodiments, the binding protein comprises a VP domain comprising an FR1 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 33, 49, 65, or 81, or an FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The insertion(s) and / or deletion(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof.

[0401] In some embodiments, the binding protein comprises a VP domain comprising an FR2 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 34, 50, 66, or 82, or an FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The insertion(s) and / or deletion(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof.

[0402] In some embodiments, the binding protein comprises a VP domain comprising an FR3 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 35, 51, 67, or 83, or an FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The insertion(s) and / or deletion(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof.

[0403] In some embodiments, the binding protein comprises a VP domain comprising an FR4 that comprises at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of any one of SEQ ID NOs: 136, 52, 68, or 84, or an FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho method from the variable domain of any one of SEQ ID NOs: 29, 45, 61, or 77. The insertion(s) and / or deletion(s) can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof.

[0404] The binding protein can comprise a TCRa FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region; a TCRP FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a combination thereof.

[0405] In some embodiments: (i) the Va domain comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or sequence similarity to the amino acid sequence set forth in SEQ ID NO.: 13, 15, 17, 19, 21, or 23; 37; 53; or 69; and / or (ii) the VP domain comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or sequence similarity to the amino acid sequence set forth in SEQ ID NO: 29; 45; 61; or 77, respectively.

[0406] The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0407] In some embodiments a binding protein is provided that is capable of binding to a KRAS G12V peptide antigen:HLA-A2 complex, wherein the peptide antigen comprises, consists essentially of, or consists of any one of SEQ ID NOs: 2, 5, 6, or 7, particularly SEQ ID NO: 2 and wherein the HLA is optionally an HLA-A*02:01. In certain embodiments, the binding protein comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NO.: 13, 15, 17, 19, 21, or 23; 37; 53; or 69 and the second polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 30; 46; 62; or 78, respectively. The first polypeptide can be or comprise a TCRa chain and / or the second polypeptide can be or comprise a TCRP chain. In some embodiments, the first polypeptide iis or comprises a TCRa chain and / or the second polypeptide is or comprises a TCRP chain.

[0408] In some embodiments a binding protein is provided that comprises a TCR a-chain and a TCR P-chain, wherein the TCR a-chain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13, 15, 17, 19, 21, or 23; 37; 53; or 69 and the TCR P-chain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 29; 45; 61; or 78.

[0409] In some embodiments, a variable domain comprises an amino acid sequence with one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69 . The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0410] For example, the variable domain can comprise an amino acid sequence with at least 1, 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid insertions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the variable domain comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid insertions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0411] In some embodiments, the variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid insertions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0412] The one or more insertions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions can be contiguous, noncontiguous, or a combination thereof.

[0413] In some embodiments, a variable domain comprises an amino acid sequence with at least 1, 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid deletions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69 . The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0414] In some embodiments, the variable domain comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid deletions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69. The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0415] In some embodiments, the variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions relative to any one of SEQ ID NOs: 13, 15, 17, 19, 21, 23, 37, 53, or 69 . The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0416] The one or more deletions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, noncontiguous, or a combination thereof.

[0417] In some embodiments, a variable domain comprises an amino acid sequence with at least 1, 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, or at least 30 amino acid substitutions relative to any one of SEQ ID NOs:

[0418] 13, 15, 17, 19, 21, and 23 . The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0419] In some embodiments, the variable domain comprises an amino acid sequence with at most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 25, at most 30, at most 35, at most 40, at most 45, or at most 50 amino acid substitutions relative to any one of SEQ ID NOs:

[0420] 13, 15, 17, 19, 21, and 23 . The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0421] In some embodiments, the variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid substitutions relative to any one of SEQ ID NOs:

[0422] 13, 15, 17, 19, 21, and 23 . The Va domain may not have a CDR3a comprising the amino acid sequence set forth in SEQ ID NO: 12. The Va domain also may not comprise the amino acid sequence set forth in SEQ ID NO: 9.

[0423] The one or more substitutions can be at the N-terminus, the C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions can be contiguous, noncontiguous, or a combination thereof.

[0424] The binding protein can further comprise a TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP). The TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP) can be human. The TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP) can be mammalian. The TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP) can be an engineered variant of a mammalian (e.g. human) constant domain. In some embodiments, the Ca is an engineered variant of a human Ca and / or the CP is an engineered variant of a human Cp. In some embodiments, the Ca is an engineered variant of a human Ca and the CP is an engineered variant of a human Cp.

[0425] The binding protein can comprise (i) an extracellular domain of a TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain; (ii) a transmembrane domain of a TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain; and / or (iii) a cytoplasmic domain of a TCR alpha chain, TCR beta chain, TCR gamma chain, or TCR delta chain. The binding protein can comprise a full length or substantially full length TCR alpha chain, TCR beta chain, TCR gamma chain, and / or TCR delta chain.

[0426] In any of the presently disclosed embodiments, a binding protein can comprise a TCR, a single-chain TCR (scTCR), a scTv, dsTv, or a chimeric antigen receptor (CAR). Methods for producing engineered TCRs are described in, for example, Bowerman et al., Mol. Immunol., 6(15):3000 (2009), the techniques of which are herein incorporated by reference. Methods for making CARs are known in the art and are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens etal., 2007, Clin. Cancer Res. 73:5426, the techniques of which are herein incorporated by reference. In some embodiments, a binding protein comprises a soluble TCR, optionally fused to a binding domain (e.g., a scFv) specific for a CD3 protein. See Elie Dolgin, Nature Biotechnology 40 AM- 449 (2022).

[0427] In any of the presently disclosed embodiments, a polynucleotide encoding a binding protein can further comprise: (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor p chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii). Without being bound by theory, in certain embodiments, coexpression or concurrent expression of a binding protein and a CD8 co-receptor protein or portion thereof functional to bind to an HLA molecule may improve one or more desired activity of a host cell (e.g., immune cell, such as a T cell, optionally a CD4+T cell) as compared to expression of the binding protein alone. It will be understood that the binding protein-encoding polynucleotide and the CD8 co-receptor polypeptide-encoding polynucleotide may be present on a single nucleic acid molecule (e.g., in a same expression vector), or may be present on separate nucleic acid molecules in a host cell.

[0428] In any of the presently disclosed embodiments, a CD8 co-receptor alpha chain can comprise, consist essentially of, or consist of SEQ ID NO.:93, or SEQ ID NO.:93 with the signal peptide removed. An example of a polynucleotide encoding SEQ ID NO.: 93 is provided in SEQ ID NO.:94. In some embodiments, a CD8 co-receptor alpha chain comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO.:93, or SEQ ID NO.:93 with the signal peptide removed.

[0429] In any of the presently disclosed embodiments, a CD8 co-receptor beta chain can comprise, consist essentially of, or consist of SEQ ID NO.:95, or SEQ ID NO.:95 with the signal peptide removed. An example of a polynucleotide encoding SEQ ID NO.: 95 is provided in SEQ ID NO.:96. In some embodiments, a CD8 co-receptor beta chain comprises, consists essentially of, or consists of an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO.:95, or SEQ ID NO.:95 with the signal peptide removed.

[0430] In certain further embodiments, a polynucleotide comprises: (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b). In further embodiments, a polynucleotide comprises a polynucleotide that encodes a self-cleaving peptide and is disposed between: (1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; and / or (2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain.

[0431] In still further embodiments, a polynucleotide can comprise, operably linked in-frame: (i) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnBP); (ii) (pnCD8P)-(pnSCPl)-(pnCD8a)- (pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCPl)-(pnCD8a)-(pnSCP2)-(pnCD8P); (iv) (pnBP)- (pnSCPl)-(pnCD8P)-(pnSCP2)-(pnCD8a); (v) (pnCD8a)-(pnSCPl)-(pnBP)-(pnSCP2)- (pnCD8P); or (vi) (pnCD8P)-(pnSCPl)-(pnBP)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnBP is the polynucleotide encoding a binding protein, and wherein pnSCPl and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different (e.g., P2A, T2A, F2A, E2A). It will be understood that self-cleaving peptide can comprise a linker N-terminal and / or C-terminal thereto. An example of a linker is GSG. In some embodiments, a T2A peptide is provided that comprises a N-terminal GSG linker.

[0432] In certain embodiments, the encoded binding protein comprises a TCRa chain and a TCRP chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain. In further embodiments, the polynucleotide comprises, operably linked in-frame: (i) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa);

[0433] (ii) (pnCD8P)-(pnSCPl)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa); (iii) (pnCD8a)- (pnSCPl)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (iv) (pnCD8P)-(pnSCPl)- (pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP); (v) (pnTCRP)-(pnSCPl)-(pnTCRa)- (pnSCP2)-(pnCD8a)-(pnSCP3)-(pnCD8P); (vi) (pnTCRP)-(pnSCPl)-(pnTCRa)-(pnSCP2)- (pnCD8P)-(pnSCP3)-(pnCD8a); (vii) (pnTCRa)-(pnSCPl)-(pnTCRP)-(pnSCP2)-(pnCD8a)- (pnSCP3)-(pnCD8P); (viii) (pnTCRa)-(pnSCPl)-(pnTCRP)-(pnSCP2)-(pnCD8P)-(pnSCP3)- (pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnTCRa is the polynucleotide encoding a TCR a chain, wherein pnTCRP is the polynucleotide encoding a TCR P chain, and wherein pnSCPl, pnSCP2, and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0434] Additionally or alternatively, a polynucleotide encoding a binding protein can encode a furin cleavage site or other protease cleavage site disposed between two other polypeptides (e.g., between a TCRP chain and a TCRa chain.

[0435] In certain embodiments, an encoded polypeptide of the present disclosure comprises one or more junction amino acids. “Junction amino acids” or “junction amino acid residues” refer to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can result from the design of a construct that encodes a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein of this disclosure (e.g., a P2A peptide disposed between a TCR a-chain and a TCR P-chain, the self-cleavage of which can leave one or more junction amino acids in the a-chain, the TCR P-chain, or both).

[0436] In further embodiments, a binding protein is expressed as part of a transgene construct that encodes, and / or a host cell of the present disclosure can encode: one or more additional accessory protein, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor P-chain; a CD8 co-receptor a-chain or both; or any combination thereof. Polynucleotides and transgene constructs useful for encoding and expressing binding proteins and accessory components (e.g., one or more of a safety switch protein, a selection marker, CD8 co-receptor P- chain, or a CD8 co-receptor a-chain) are described in PCT application PCT / US2017 / 053112, the polynucleotides, transgene constructs, and accessory components, including the nucleotide and amino acid sequences, of which are hereby incorporated by reference. It will be understood that any or all of a binding protein of the present disclosure, a safety switch protein, a tag, a selection marker, a CD8 co-receptor P-chain, or a CD8 co-receptor a-chain may be encoded by a single nucleic acid molecule or may be encoded by polynucleotide sequences that are, or are present on, separate nucleic acid molecules.

[0437] Exemplary safety switch proteins include, for example, a truncated EGF receptor polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity, but that retains its native amino acid sequence, has type I transmembrane cell surface localization, and has a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118: 1255-1263, 2011); a caspase polypeptide (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; Di Stasi et al., N. Engl. J. Med. 365: 1673-1683, 2011; Zhou and Brenner, Exp. Hematol. pii:S0301-472X(16)30513-6. doi : 10.1016 / j . exphem.2016.07. Oi l), RQR8 (Philip et al., Blood 124: 1277-1287, 2014); a 10-amino-acid tag derived from the human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Set. USA 105:623-628, 2008); and a marker / safety switch polypeptide, such as RQR (CD20 + CD34; Philip et al., 2014). Other accessory components useful for modified host cells of the present disclosure comprise a tag or selection marker that allows the cells to be identified, sorted, isolated, enriched, or tracked. For example, marked host cells having desired characteristics (e.g., an antigen-specific TCR and a safety switch protein) can be sorted away from unmarked cells in a sample and more efficiently activated and expanded for inclusion in a product of desired purity.

[0438] As used herein, the term “selection marker” comprises a nucleic acid construct (and the encoded gene product) that confers an identifiable change to a cell permitting detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker. RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, an RQR-encoding polynucleotide comprises a polynucleotide that encodes the 16-amino-acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is incorporated at the amino terminal position of a CD8 co-receptor stalk domain (Q8). In further embodiments, the CD34 minimal binding site sequence can be combined with a target epitope for CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated by reference herein). This construct allows for the selection of host cells expressing the construct, with for example, CD34 specific antibody bound to magnetic beads (Miltenyi) and that utilizes clinically accepted pharmaceutical antibody, rituximab, that allows for the selective deletion of a transgene expressing engineered T cell (Philip et al., 2014).

[0439] Further exemplary selection markers also include several truncated type I transmembrane proteins normally not expressed on T cells: the truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (see for example, Di Stasi et al., N Engl. J. Med. 365: 1673-1683, 2011; Mavilio et al., Blood 83:1988-1991, 1994; Fehse et al., Mol. Ther. 7:448- 456, 2000; each incorporated herein in their entirety). A useful feature of CD 19 and CD34 is the availability of the off-the-shelf Miltenyi CliniMACs™ selection system that can target these markers for clinical-grade sorting. However, CD 19 and CD34 are relatively large surface proteins that may tax the vector packaging capacity and transcriptional efficiency of an integrating vector. Surface markers containing the extracellular, non-signaling domains or various proteins (e.g., CD 19, CD34, LNGFR) also can be employed. Any selection marker may be employed and should be acceptable for Good Manufacturing Practices. In certain embodiments, selection markers are expressed with a polynucleotide that encodes a gene product of interest (e.g., a binding protein of the present disclosure, such as a TCR or CAR). Further examples of selection markers include, for example, reporters such as GFP, EGFP, P-gal or chloramphenicol acetyltransferase (CAT). In certain embodiments, a selection marker, such as, for example, CD34 is expressed by a cell and the CD34 can be used to select enrich for, or isolate (e.g., by immunomagnetic selection) the transduced cells of interest for use in the methods described herein. As used herein, a CD34 marker is distinguished from an anti-CD34 antibody, or, for example, a scFv, TCR, or another antigen recognition moiety that binds to CD34.

[0440] In certain embodiments, a selection marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD 19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

[0441] Regarding RQR polypeptides, without wishing to be bound by theory, it is believed that distance from the host cell surface is important for RQR polypeptides to function as selection markers / safety switches (Philip etal., 2010 (supra)). In some embodiments, the encoded RQR polypeptide is contained in a P-chain, an a-chain, or both, or a fragment or variant of either or both, of the encoded CD8 co-receptor. In specific embodiments, a modified host cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein that comprises a P-chain containing a RQR polypeptide and further comprises a CD8 a-chain.

[0442] An encoded CD8 co-receptor includes, in some embodiments, an a-chain or a fragment or variant thereof. An amino acid sequence of the human CD8 co-receptor a -chain precursor is known and is provided at, for example, UniProtKB -P30433 (see also UniProtKB - P31783; - P10732; and -P10731). An encoded CD8 co-receptor includes, in some embodiments, a P-chain or a fragment or variant thereof. An amino acid sequence of the human CD8 co-receptor P-chain precursor is known and is provided at, for example, UniProtKB -Pl 0966 (see also UniProtKB - Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).

[0443] An isolated polynucleotide of this disclosure may further comprise a polynucleotide encoding a safety switch protein, a selection marker, a CD8 co-receptor beta chain, or a CD8 co- receptor alpha chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.

[0444] In any of the presently disclosed embodiments, a polynucleotide can be codon optimized for expression in a host cell. In some embodiments, the host cell comprises a human immune system cell, such as a T cell, a NK cell, or a NK-T cell (Scholten et al.. Clin. Immunol. 119: 135, 2006). Codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimumGene™ tool, or GeneArt (Life Technologies). Codon-optimized sequences include sequences that are partially codon-optimized (z.e., one or more of the codons is optimized for expression in the host cell) and those that are fully codon-optimized. It will be appreciated that in embodiments wherein a polynucleotide encodes more than one polypeptide (e.g., a TCR a chain, a TCR P chain, a CD8 co-receptor a chain, a CD8 co-receptor p chain, and one or more self-cleaving peptides), each polypeptide can independently fully codon optimized, partially codon optimized, or not codon optimized.

[0445] In another aspect, the present disclosure provides an expression vector, comprising any polynucleotide as provided herein operably linked to an expression control sequence.

[0446] Also provided herein are vectors that comprise a polynucleotide or transgene construct of the instant disclosure. Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector, retroviral vector). Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as “expression vectors”). According to related embodiments, it is further understood that, if one or more agents (e.g., polynucleotides encoding polypeptides as described herein) are co administered to a subject, that each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.

[0447] In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may 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 (z.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0448] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from lentiviral vector or a y-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0449] “Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome. “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses. “Lentiviral vector,” as used herein, means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells. In some embodiments, a lentiviral vector is a selfinactivating lentiviral vector. A self-inactivating lentiviral vector can comprise a modification to prevent the transfer of enhancer and promoter elements in the 5' long terminal repeat (LTR) of the vector to transduced cells, for example, comprising a deletion in the 3'LTR of the viral genome that is transferred into the 5 'LTR after one round of reverse transcription, resulting in a provirus that contains no LTR derived enhancer or promoter elements. In some embodiments, a lentiviral vector is a third generation lentiviral vector. A third generation lentiviral vector can utilize a packaging system split into two or more plasmids, e.g., one encoding Rev and one encoding Gag and Pol. A third generation lentiviral vector can utilize a packaging system that lacks Tat or does not require Tat expression, and instead comprises, e.g., a chimeric 5' LTR fused to a heterologous promoter on the transfer plasmid.

[0450] In certain embodiments, the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV- 1 -derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing TCR or CAR transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6:321939, 2011; Zhao et al., J. Immunol. 774:4415, 2005; Engels et al., Hum. Gene Ther. 74: 1155, 2003; Frecha et al., Mol. Ther. 18: 1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:91, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)- based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).

[0451] Other vectors developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a- viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0452] When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi ci str onic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

[0453] In certain embodiments, a vector is capable of delivering the polynucleotide or transgene construct to a host cell (e.g., a hematopoietic progenitor cell or a human immune system cell). In specific embodiments, a vector is capable of delivering a polynucleotide or transgene construct to human immune system cell, such as, for example, a CD4+T cell, a CD8+T cell, a CD4' CD8' double negative T cell, a stem cell memory T cell, a y5 T cell, a natural killer cell, a dendritic cell, or any combination thereof. In further embodiments, a vector is capable of delivering a transgene construct to a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. In some embodiments, a vector that encodes a polynucleotide or transgene construct of the present disclosure may further comprise a polynucleotide that encodes a nuclease that can be used to perform a chromosomal knockout in a host cell (e.g., a CRISPR-Cas endonuclease or another endonuclease as disclosed herein) or that can be used to deliver a therapeutic polynucleotide or transgene or portion thereof to a host cell in a gene therapy replacement or gene repair therapy. Alternatively, a nuclease used for a chromosomal knockout or a gene replacement or gene repair therapy can be delivered to a host cell independent of a vector that encodes a polynucleotide or transgene construct of this disclosure.

[0454] In certain embodiments, the vector is capable of delivering the polynucleotide to a host cell. In further embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In still further embodiments, the human immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof. In yet further embodiments, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0455] In any of the presently disclosed embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector or a y-retroviral vector.

[0456] Examples of transposon-based systems that can be used include, but are not limited to, sleeping beauty (e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON.

[0457] Also provided herein are host cells that encode and / or express a binding protein (and, optionally, one or more accessory protein, such as a transduction marker, a CD8 co-receptor polypeptide, or the like, as provided herein). In certain embodiments, a host cell is provided that is modified to comprise a polynucleotide and / or an expression vector of the present disclosure, and / or to express a binding protein of the present disclosure.

[0458] Any suitable host cell may be modified to include a heterologous polynucleotide encoding a binding protein of this disclosure, including, for example, an immune cell, such as T cell, a NK cell, or a NK-T cell modified to include the heterologous polynucleotide. In some embodiments, a modified immune cell comprises a CD4+T cell, a CD8+T cell, or both. Methods for transfecting / transducing T cells with desired nucleic acids have been described e.g., U.S. Patent Application Pub. No. US 2004 / 0087025) as have adoptive transfer procedures using T cells of desired target-specificity e.g., Schmitt et al., Hum. Gen. 20: 1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther.

[0459] 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011 / 0243972; US 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), such that adaptation of these methodologies to the presently disclosed embodiments is contemplated, based on the teachings herein.

[0460] Any appropriate method can be used to transfect or transduce the cells, for example, the T cells, or to administer the polynucleotides or compositions of the present methods. Known methods for delivering polynucleotides to host cells include, for example, use of cationic polymers, lipid-like molecules, and certain commercial products such as, for example, IN-VIVO- JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and the like. Still further methods of transfecting or transducing host cells employ vectors, described in further detail herein.

[0461] In some embodiments, the polynucleotide, expression construct, or targeting polynucleotide construct is comprised in a carrier of formulation, wherein the carrier or formulation comprises a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), a lipopolyplex (LPP), a cationic polypeptide, a polymeric nanoparticle, or a nanoscale platform, such as a nanoemulsion.

[0462] In certain embodiments, the host cell or modified cell comprises a hematopoietic progenitor cell, stem cell (e.g., iPSC), and / or or human immune cell. In some embodiments, the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof. In further embodiments, the immune cell comprises a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a y5 T cell, or any combination thereof. In certain further embodiments, the immune cell comprises a CD4+ T cell and a CD8+ T cell. In certain still further embodiments, the CD4+ T cell, the CD8+ T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor p chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii). A host cell can be a peripheral blood mononuclear cell (PBMC). A host cell can be a lymphoid cell. A host cell can be a lymphocyte. A host cell can be a T cell. A host cell can be an alpha beta T cell (whether expressing or not expressing an endogenous alpha-beta TCR). A host cell can be a gamma delta T cell (whether expressing or not expressing an endogenous gammadelta TCR). A host cell can be a B cell. A host cell can be a natural killer (NK) cell. A host cell can be a Natural Killer T (NKT) cell. A host cell can be a mammalian cell. A host cell can be a human cell.

[0463] A host cell can be a primary cell. A host cell can be an immortalized cell. A host cell can be of a cell line. A host cell can be differentiated from a stem cell, for example, an induced pluripotent stem cell (iPSC), embryonic stem cell, hematopoietic stem cell (HSC), or the like.

[0464] In any of the foregoing embodiments, a host cell (e.g., an immune cell) may modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide involved in immune signaling or other related activities. Exemplary gene knockouts include those that encode PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA molecule, a TCR molecule, or the like. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may be recognized as foreign by an allogeneic host receiving the modified immune cells, which may result in elimination of the modified immune cells (e.g., an HLA allele), or may downregulate the immune activity of the modified immune cells (e.g, PD-1, LAG-3, CTLA4, FasL, TIGIT, TIM3), or may interfere with the binding activity of a heterologously expressed binding protein of the present disclosure (e.g, an endogenous TCR of a modified T cell that binds a non-Ras antigen and thereby interferes with the modified immune cell binding a cell that expresses a Ras antigen).

[0465] Accordingly, decreasing or eliminating expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of the modified cells in an autologous or allogeneic host setting, and may allow for universal administration of the cells (e.g., to any recipient regardless of HLA type). In certain embodiments, a modified cell is a donor cell (e.g., allogeneic) or an autologous cell. In certain embodiments, a modified cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA component (e.g., a gene that encodes an al macroglobulin, an a2 macroglobulin, an a3 macroglobulin, a pi microglobulin, or a P2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai el al., Blood 779(24):5697 (2012); and Torikai et al., Blood 722(8): 1341 (2013), the gene-editing techniques, compositions, and adoptive cell therapies of which are herein incorporated by reference in their entirety).

[0466] As used herein, the term “chromosomal gene knockout” refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0467] In certain embodiments, a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell. Chromosomal editing can be performed using, for example, endonucleases. As used herein “endonuclease” refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain. In certain embodiments, an endonuclease is capable of cleaving a targeted gene thereby inactivating or “knocking out” the targeted gene. An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease. The nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non- homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule may be used for a donor gene “knock-in”, for target gene “knock-out”, and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event. NHEJ is an error-prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide. NHEJ may be used to “knock-out” a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0468] As used herein, a “zinc finger nuclease” (ZFN) refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285: 1917-1934, 1999). Multiple zinc finger motifs can be linked in tandem to create binding specificity to desired DNA sequences, such as regions having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair. Alternatively, a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.

[0469] As used herein, a “transcription activator-like effector nuclease” (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA cleavage domain, such as a FokI endonuclease. A “TALE DNA binding domain” or “TALE” is composed of one or more TALE repeat domains / units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids. The TALE repeat domains are involved in binding of the TALE to a target DNA sequence. The divergent amino acid residues, referred to as the Repeat Variable Diresidue (RVD), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagineisoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagineglycine) binds to a T nucleotide. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. US 2011 / 0301073, which atypical RVDs are incorporated by reference herein in their entirety). TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells. Non- homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.

[0470] As used herein, a “clustered regularly interspaced short palindromic repeats / Cas” (CRISPR / Cas) nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence. CRISPR / Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases. The crRNA-guided surveillance complexes in types I and III need multiple Cas subunits. Type II system, the most studied, comprises at least three components: an RNA- guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA comprises a duplex forming region. A crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM. Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and / or deletions which disrupt expression of the targeted locus. Alternatively, a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair. The crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012). Further, the region of the guide RNA complementary to the target site can be altered or programed to target a desired sequence (Xie et al., PLOS One 9:el00448, 2014; U.S. Pat. Appl. Pub. No. US 2014 / 0068797, U.S. Pat. Appl. Pub. No. US 2014 / 0186843; U.S. Pat. No. 8,697,359, and PCT Publication No. WO 2015 / 071474; each of which is incorporated by reference). In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR / Cas nuclease system.

[0471] Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.

[0472] As used herein, a “meganuclease,” also referred to as a “homing endonuclease,” refers to an endodeoxyribonuclease characterized by a large recognition site (double stranded DNA sequences of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structure motifs: LAGLID ADG, GIY-YIG, HNH, His-Cys box and PD- (D / E)XK. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, Pl-Sce, 1-SceIV, I-CsmI, I- Panl, I-Scell, I-Ppol, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res . 25:3379-3388, 1997; Dujon et al., Gene 82:115-118, 1989; Perl er et al., Nucleic Acids Res. 22: 1125-1127, 1994; Jasin, Trends Genet. 72:224-228, 1996; Gimble et al., J. Mol. Biol. 263: 163-180, 1996; Argast et al., J. Mol. Biol. 250:345-353, 1998).

[0473] In certain embodiments, naturally occurring meganucleases may be used to promote sitespecific genome modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, an HLA-encoding gene, or a TCR component-encoding gene. In other embodiments, an engineered meganuclease having a novel binding specificity for a target gene is used for sitespecific genome modification (see, e.g., Porteus et al., Nat. BiotechnoL 23'961-13, 2005; Sussman et al., J. Mol. Biol. 342.31-41, 2004; Epinat et al., Nucleic Acids Res. 31 :2952-62, 2003; Chevalier et al., Molec. Cell 70:895-905, 2002; Ashworth et al., Nature 441.656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, 2007; U.S. Patent Publication Nos. US 2007 / 0117128; US 2006 / 0206949; US 2006 / 0153826; US 2006 / 0078552; and US 2004 / 0002092). In further embodiments, a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL. MegaTALs can be utilized to not only knockout one or more target genes, but to also introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

[0474] In certain embodiments, a chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g., an immune cell) comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor and wherein the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, an HL A component, or a TCR component, or any combination thereof) in the host cell.

[0475] In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR / Cas nuclease system or base editing system (Komor, A. C.; Kim, Y. B.; Packer, M. S.; Zuris, J. A.; Liu, D. R. Nature 533, 420-424 (2016). Briefly, base editing is a genome-editing approach that uses components from CRISPR systems together with other enzymes to directly introduce point mutations into cellular DNA or RNA without making double-stranded DNA breaks. Certain DNA base editors comprise a catalytically disabled nuclease fused to a nucleobase deaminase enzyme and, in some cases, a DNA glycosylase inhibitor. RNA base editors function similarly, using components that target RNA. Base editors directly convert one base or base pair into another, enabling the efficient installation of point mutations in non-dividing cells without generating excess undesired editing byproducts. See e.g., Rees H et al. Nature Reviews Genetics (2018).

[0476] A chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent. Chromosomal gene knockouts can also be inferred from the absence of gene expression (e.g., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout.

[0477] In certain embodiments, a chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.

[0478] In certain embodiments, a chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.

[0479] In some embodiments, a population of host cells comprising a binding protein disclosed herein exhibits at least 5%, 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 2-fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 60 fold, at least 70 fold, at least 80 fold, at least 90 fold, at least 100 fold, at least 150 fold, at least 200 fold, at least 250 fold, at least 300 fold, at least 350 fold, at least 400 fold, at least 500 fold, at least 600 fold, at least 700 fold, at least 800 fold, at least 900 fold, at least 1000 fold, or at least 5000 fold increased functional avidity for a target antigen of the binding protein as compared to a population of control cells (for example, cells expressing a control binding protein specific for the same target antigen). The host cells can comprise a binding protein (e.g., a TCR comprising Va and VP regions and / or CDRs disclosed herein) that binds a target antigen (for example, a KRAS G12 mutant peptide, such as KRAS G12V mutant peptide, e.g., present in a peptide:HLA complex). The increase in avidity can be, for example, as determined by an assay for determining expression an activation marker (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, a cytokine, an interleukin, an interferon) upon exposure to target cells that express or present the target antigen, or and / or an assay to determine EC50 (e.g., peptide dose at which a half-maximal activation of a T cell population is reached). In some embodiments, the host cells and the control cells are both T cells, and the host cell and control cell populations can comprise the same, about the same, or substantially the same composition or amount(s) of T cell type(s) (e.g., CD4+, CD8+, or both).

[0480] In some embodiments, a population of host cells comprising a binding protein disclosed herein exhibits at least 5%, at least 10%, at least 20%, at least 30%, a...

Claims

CLAIMSWhat is claimed is:

1. A binding protein that is capable of binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV (SEQ ID NO:2) and the HLA is HLA- A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing 2 or 3 methyl groups.

2. The binding protein of claim 1, wherein the N-terminal lysine of the peptide comprises a side chain bearing 2 methyl groups.

3. The binding protein of claim 1, wherein the N-terminal lysine of the peptide comprises a side chain bearing 3 methyl groups.

4. The binding protein of any one of claims 1-3, wherein the binding protein is also capable of binding to peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV (SEQ ID NO:2) and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing 0 or 1 methyl groups.

5. The binding protein of any one of claims 1-4, wherein the binding protein comprises a binding domain that binds the peptide:HLA complex, wherein the binding domain optionally comprises an immunoglobulin superfamily variable domain.

6. The binding protein of claim 5, wherein binding domain comprises a TCR a-chain variable domain (Va) and / or a TCR P-chain variable domain (VP), wherein the binding domain optionally comprises the Va domain and the VP domain.

7. The binding protein of any one of claims 1-6, comprising a heavy chain variable domain (VH) and / or a light chain variable domain (VL) of a TCR-mimic antibody, wherein the binding domain optionally comprises the VH domain and the VL domain.

8. A binding protein comprising a TCR a-chain variable domain (Va) and a TCR P- chain variable domain (VP), wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV (SEQ ID NO:2), the N-terminal lysine of the peptide bears zero, one, two, or three methyl groups, and the HLA is HLA-A*02:01, and wherein the Va domain comprises complementarity determining region (CDR)la, CDR2a, and CDR3a amino acid sequences and the VP domain comprises CDRip, CDR2P, and CDR3P amino acid sequences, wherein the CDRla, CDR2a, CDR3a CDRip, CDR2P, and CDR3P amino acid sequences are:(i) DSSSTY (SEQ ID NO: 10), IFSNMDM (SEQ ID NO: 11), CAEIEDRTDKLIF (SEQ ID NO:24), SGHAT (SEQ ID NO:30), FQDESV (SEQ ID NO:31), and CASSPTGEGEQYF (SEQ ID NO:32), respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRT, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRT, as shown in Table Z;(ii) DSSSTY, IFSNMDM, CAEIEDSTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDST, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDST, as shown in Table Z;(iii) DSSSTY, IFSNMDM, CAEIEDRSDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRS, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRS, as shown in Table Z;(iv) DSSSTY, IFSNMDM, CAEITGNTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR TGNT, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR TGNT, as shown in Table Z;(v) DSSSTY, IFSNMDM, CAEIEDVADKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDVA, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDVA, as shown in Table Z;(vi) DSSSTY, IFSNMDM, CAEIEARRDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EARR, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EARR, as shown in Table Z;(vii) YGATPY, YFSGDTLV, CAVGADSNYQLIW, SGHNS, FNNNVP, and CASSLVFYNEQFF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-l, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acidsequence identity to the VP domain amino acid sequence of TCR A2UoMl-l, as shown in Table Z;(viii) NSAFQY, TYSSGN, CAMSDDWGKLQF, PRHDT, FYEKMQ, and CASSTIAGGRGNEQFF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-2, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-2, as shown in Table Z; or(ix) DSAIYN, IQSSQRE, CAVPPGDDYKLSF, MDHEN, SYDVKM, and CASSPIQRGDQPQHF, respectively, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-4, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-4, as shown in Table Z.

9. The binding protein of claim 8, wherein the CDRla, CDR2a, CDR3a CDRip, CDR2P, and CDR3P amino acid sequences are:(i) DSSSTY, IFSNMDM, CAEIEDRTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRT, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRT, as shown in Table Z;(ii) DSSSTY, IFSNMDM, CAEIEDSTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein: the Va domain comprises an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDST, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDST, as shown in Table Z;(iii) DSSSTY, IFSNMDM, CAEIEDRSDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRS, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRS, as shown in Table Z;(iv) DSSSTY, IFSNMDM, CAEITGNTDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR TGNT, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR TGNT, as shown in Table Z;(v) DSSSTY, IFSNMDM, CAEIEDVADKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDVA, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDVA, as shown in Table Z;(vi) DSSSTY, IFSNMDM, CAEIEARRDKLIF, SGHAT, FQDESV, and CASSPTGEGEQYF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EARR, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EARR, as shown in Table Z;(vii) YGATPY, YFSGDTLV, CAVGADSNYQLIW, SGHNS, FNNNVP, and CASSLVFYNEQFF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-l, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-l, as shown in Table Z;(viii) NSAFQY, TYSSGN, CAMSDDWGKLQF, PRHDT, FYEKMQ, and CASSTIAGGRGNEQFF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-2, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-2, as shown in Table Z; or(ix) DSAIYN, IQSSQRE, CAVPPGDDYKLSF, MDHEN, SYDVKM, and CASSPIQRGDQPQHF, respectively, wherein: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-4, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR A2UoMl-4, as shown in Table Z.

10. A T cell receptor comprising an alpha chain and a beta chain, wherein:the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDRT (4UM), as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDRT (4UM), as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRT, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR EDRT, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDST, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDST, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDST, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDST, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDRS, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDRS, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDRS, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDRS, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR TGNT, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR TGNT, as shown in Table X, wherein, optionally: the Va domain comprisesan amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR TGNT, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR TGNT, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EDVA, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EDVA, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EDVA, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EDVA, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR EARR, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR EARR, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR EARR, as shown in Table Y; and the VP domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the VP domain amino acid sequence of TCR EARR, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-l, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-l, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-l, as shown in TableY; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR A2UoMl-l, as shown in Table Z; the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-2, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-2, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-2, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR A2UoMl-2, as shown in Table Z; or the alpha chain comprises in a variable domain the three alpha chain CDRs of TCR A2UoM-l-4, as shown in Table X, and the beta chain comprises in a variable domain the three beta chain CDRs of TCR A2UoM-l-4, as shown in Table X, wherein, optionally: the Va domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the Va domain amino acid sequence of TCR A2UoMl-4, as shown in Table Y; and the V|3 domain comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the V|3 domain amino acid sequence of TCR A2UoMl-4, as shown in Table Z.

11. A T cell receptor comprising an alpha chain and a beta chain, wherein: the alpha chain comprises the alpha chain variable domain TCR EDRT (4UM), as shown in Table Y, and the beta chain variable domain of TCR EDRT (4UM), as shown in Table Z; the alpha chain comprises the alpha chain variable domain TCR EDST, as shown in Table Y, and the beta chain variable domain of TCR EDST, as shown in Table Z; the alpha chain comprises the alpha chain variable domain TCR EDRS, as shown in Table Y, and the beta chain variable domain of TCR EDRS (4UM) or of TCR EDRS, as shown in Table Z;the alpha chain comprises the alpha chain variable domain TCR TGNT, as shown in Table Y, and the beta chain variable domain of TCR TGNT, as shown in Table Z; the alpha chain comprises the alpha chain variable domain TCR EDVA, as shown in Table Y, and the beta chain variable domain of TCR EDVA, as shown in Table Z; the alpha chain comprises the alpha chain variable domain TCR EARR, as shown in Table Y, and the beta chain variable domain of TCR EARR, as shown in Table Z; the alpha chain comprises the alpha chain variable domain TCR A2UoM-l-l, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-l, as shown in Table Z; the alpha chain comprises the alpha chain variable domain TCR A2UoM-l-2, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-2, as shown in Table Z; or the alpha chain comprises the alpha chain variable domain TCR A2UoM-l-4, as shown in Table Y, and the beta chain variable domain of TCR A2UoM-l-4, as shown in Table Z.

12. A binding protein comprising a TCR alpha chain variable domain of any one of the TCRs shown in Table Y and the corresponding beta chain variable domain shown in Table X.

13. A binding protein comprising a TCR alpha chain variable domain (Va) and a TCR beta chain variable domain (VP), wherein the Va and the VP comprise the amino acid sequences:EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQR LTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDRTDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLP KDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively;EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQR LTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDSTDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively;EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDRSDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively;EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEITGNTDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively;EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDVADKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively;EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEARRDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively;QSVTQPDIHITVSEGASLELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRKPSVHWSDAAEYFCAVGADSNYQLIWGAGTKLIIKPD andGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLVFYNEQFFGPGTRLTVLE , respectively;QKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDDWGKLQFGAGTQVVVTPD andGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPD RFSAQQFSDYHSELNMSSLELGDSALYFCASSTIAGGRGNEQFFGPGTRLTVLE , respectively; orKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSG RLNASLDKSSGRSTLYIAASQPGDSATYLCAVPPGDDYKLSFGAGTTVTVRAN andSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGY SVSREKKERFSLILESASTNQTSMYLCASSPIQRGDQPQHFGDGTRLSILE , respectively.

14. The binding protein of claim 13, wherein the binding protein comprises: a TCR comprising an alpha chain and a beta chain; a single-chain TCR; a single-chain TCR variable fragment (scTv); a disulfide stabilized TCR variable fragment (dsFv); or a TCR / CAR.

15. A TCR comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise the following amino acid sequence disposed N-terminal to the alpha chain constant domain and the beta chain constant domain, respectively:EDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQR LTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDRTDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLP KDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively; orEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQR LTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDSTDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLP KDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively; orEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQR LTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDRSDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively; orEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEITGNTDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively; orEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEDVADKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively; orEDVEQSLFLSVREGDSSVINCTYTDSSSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYFCAEIEARRDKLIFGTGTRLQVFPN andEVAQSPRYKITEKSQAVAFWCDPISGHATLYWYRQILGQGPELLVQFQDESVVDDSQLPKDRFSAERLKGVDSTLKIQPAELGDSAMYLCASSPTGEGEQYFGPGTRLTVT , respectively; orQSVTQPDIHITVSEGASLELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRKPSVHWSDAAEYFCAVGADSNYQLIWGAGTKLIIKPD andGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLVFYNEQFFGPGTRLTVLE , respectively; orQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDDWGKLQFGAGTQVVVTPD andGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSTIAGGRGNEQFFGPGTRLTVLE , respectively; orKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVPPGDDYKLSFGAGTTVTVRAN andSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGY SVSREKKERFSLILESASTNQTSMYLCASSPIQRGDQPQHFGDGTRLSILE , respectively.

16. The binding protein of any one of claims 1-13, further comprising a TCR a chain constant domain (Ca) and / or a TCR P chain constant domain (CP), optionally comprising a Ca and a C .

17. The binding protein of claim 12, 13, 14, or 16, or the T cell receptor of claim 10, 11, or 15, wherein the Ca comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, one of the following amino acid sequences:IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS;IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKCVLD MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS;IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKV AGFNLLMTLRLWSS .

18. The binding protein of claim 12, 13, 14, 16, or 17, or the T cell receptor of claim 10, 11, 15, 16, or 17, wherein the CP comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, one of the following the amino acid sequences:DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQDRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDF;DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDP QPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAE AWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF;DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKEVHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQDRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG;DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKEVHSGVCTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG.

19. The binding protein or T cell receptor of any one of claims 10-18, wherein the Ca and the Cp comprise or consist of an engineered variant of a human Ca and an engineered variant of a human CP, respectively.

20. The binding protein or T cell receptor of any one of claims 10-19, wherein the Ca, the CP, or both comprise modification(s) that promote preferential pairing of the Ca to the Cp.

21. The binding protein or T cell receptor of any one of claims 10-19, wherein the Ca and the CP each comprises an introduced cysteine residue that promotes preferential pairing of the Ca to the Cp.

22. The binding protein or T cell receptor of any one of claims 10-19, wherein the Ca comprises a T48C substitution and the CP comprises a S57C substitution to promote preferential pairing of the Ca to the Cp.

23. The binding protein or T cell receptor of any one of claims 1-22, consisting essentially of a TCR a chain and a TCR P chain.

24. The binding protein or T cell receptor of any one of claims 1-23, wherein the binding protein comprises a TCR, a single-chain TCR (scTCR), a single-chain T cell receptor variable fragment (scTv), disulfide-stabilized T cell receptor variable fragment (scTv), or a chimeric antigen receptor (CAR).

25. The binding protein of claim 24, wherein the binding protein comprises a TCR.

26. The binding protein of any one of claims 1-25, wherein the binding protein comprises an EC50 of at most 100 nM, at most 50 nM, at most 25 nM, at most 10 nM, at most 1 nM, at most 750 pM, at most 500 pM, at most 250 pM, at most 100 pM, at most 75 pM, or at most 60 pM in a CD137 surface expression assay for functional avidity to the peptide.

27. An isolated polynucleotide encoding the binding protein or T cell receptor of any one of claims 1-26.

28. An isolated polynucleotide enoding a Va domain, a VP domain, a TCR a-chain, and / or a TCR P-chain of the binding protein or T cell receptor of any one of claims 1-27.

29. The polynucleotide of claim 27 or 28, further comprising:(i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor p chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

30. The polynucleotide of claim 29, comprising:(a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor p chain; and (c) a polynucleotide encoding a selfcleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).

31. The polynucleotide of claim 29 or 30, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between:(1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; and / or(2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain.

32. The polynucleotide of any one of claims 29-31, comprising, operably linked inframe:(i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnBP);(ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnBP);(iii) (pnBP)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnCD8P);(iv) (pnBP)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnCD8a);(v) (pnCD8a)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8P); or(vi) (pnCD8P)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnBP is the polynucleotide encoding a binding protein, and wherein pnSCPi and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

33. The polynucleotide of any one of claims 29-32, wherein the encoded binding protein comprises a TCRa chain and a TCRP chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRa chain and the polynucleotide encoding a TCRP chain.

34. The polynucleotide of claim 33, comprising, operably linked in-frame:(i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa);(ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)-(pnTCRa);(iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP);(iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)-(pnTCRP);(v) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)-(pnCD8a)-(pnSCP3)-(pnCD8P);(vi) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)-(pnCD8P)-(pnSCP3)-(pnCD8a);(vii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8a)-(pnSCP3)-(pnCD8P);(viii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8P)-(pnSCP3)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor a chain, wherein pnTCRa is the polynucleotide encoding a TCR a chain, wherein pnTCRP is the polynucleotide encoding a TCR P chain, and wherein pnSCPi, pnSCP2, and pnSCPs are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

35. The polynucleotide of claim 34, wherein the pnSCPi encodes a T2A peptide, the pnSCP2 encodes a P2A peptide, and the pnSCP3 encodes a P2A peptide.

36. A polynucleotide encoding any one of the following amino acid sequences:AGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPID DSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLVFYNEQFFGPGTRLTVLEDL KNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSR GGSGATNFSLLKQAGDVEENPGPMLLELIPLLGIHFVLRTARAQSVTQPDIHITVSEGASL ELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRK PSVHWSDAAEYFCAVGADSNYQLIWGAGTKLIIKPDIQNPDPARH;MLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQ FLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSTIAGGRGNEQF FGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNG KEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSEND EWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSAL VLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGPMMKSLRVLLVILWLQLSWVWS QQKEVEQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFTAQVDKSSKYISLFIRDSQPSDSATYLCAMSDDWGKLQFGAGTQVVVTPDIQNPDPARH;VI<VTQSSRYLVI<RTGEI<VFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVI<MKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASSPIQRGDQPQHFGDGTRLSIL EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DFGSGATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEG ENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYI AASQPGDSATYLCAVPPGDDYKLSFGAGTTVTVRANIQNPDPARH.

37. A polynucleotide encoding an amino acid sequence comprisingAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSLVFYNEQFFGPGTRLTVLEDL KNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQP LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSR GGSGATNFSLLKQAGDVEENPGPMLLELIPLLGIHFVLRTARAQSVTQPDIHITVSEGASLELRCNYSYGATPYLFWYVQSPGQGLQLLLKYFSGDTLVQGIKGFEAEFKRSQSSFNLRK PSVHWSDAAEYFCAVGADSNYQLIWGAGTKLIIKPDIQNPDPARH.

38. A polynucleotide encoding an amino acid sequence comprisingMLCLLGAVSVAAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASSTIAGGRGNEQFFGPG TRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVH SGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRGGSGATNFSLLKQAGDVEENPGPMMKSLRVLLVILWLQLSWVWSQQKEV EQDPGPLSVPEGAIVSLNCTYSNSAFQYFMWYRQYSRKGPELLMYTYSSGNKEDGRFT AQVDKSSKYISLFIRDSQPSDSATYLCAMSDDWGKLQFGAGTQVVVTPDIQNPDPARH.

39. A polynucleotide encoding an amino acid sequence comprisingVI<VTQSSRYLVI<RTGEI<VFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVI<MI<EI<GDIPEGYS VSREKKERF SLILES ASTNQTSMYLC AS SPIQRGDQPQHFGDGTRLSILEDLNK VFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLK EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSG ATNFSLLKQAGDVEENPGPMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVL NCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQP GDSATYLCAVPPGDDYKLSFGAGTTVTVRANIQNPDPARH.

40. An expression vector, comprising a polynucleotide of any one of claims 27-39 operably linked to an expression control sequence.

41. The expression vector of claim 40, wherein the expression control sequence comprises an MSCV promoter.

42. The expression vector of claim 40 or claim 41, wherein the expression control sequence drives expression of a single mRNA encoding an extracellular portion of a CD8 coreceptor a chain, an extracellular portion of an CD8 co-receptor P chain, the TCR a chain, and the TCR P chain.

43. The expression vector of any one of claims 40-42, wherein the vector is capable of delivering the polynucleotide to a host cell.

44. The expression vector of claim 43, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

45. The expression vector of claim 44, wherein the human immune system cell is a CD4+T cell, a CD8+T cell, a CD4 CD8' double negative T cell, a y5 T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof.

46. The expression vector of claim 45, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

47. The expression vector of any one of claims 40-46, wherein the vector is a viral vector.

48. The expression vector of claim 47, wherein the viral vector is a lentiviral vector or a y-retroviral vector.

49. The expression vector of claim 47, wherein the viral vector is a self-inactivating lentiviral vector.

50. The expression vector of claim 47 or claim 49, wherein the viral vector is a third generation lentiviral vector.

51. A means for binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing two methyl groups.

52. A means for binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing three methyl groups.

53. A means for binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing one, two, or three methyl groups.

54. A means for binding a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and the HLA is HLA-A*02:01, and the N-terminal lysine of the peptide comprises a side chain bearing two or three methyl groups.

55. A host cell modified to comprise the polynucleotide of any one of claims 27-39 and / or the expression vector of any one of claims 40-50 and / or to express the binding protein of any one of claims 1-26 wherein, optionally, the host cell is a T cell, further optionally a human T cell.

56. A host cell expressing the binding protein of any one of claims 1-26 wherein, optionally, the host cell is a T cell, further optionally a human T cell.

57. A host cell comprising the polynucleotide of any one of claims 27-39, wherein, optionally, the polynucleotide is heterologous to the host cell wherein, further optionally, the host cell is a T cell, further optionally a human T cell.

58. A host cell comprising the expression vector of any one of claims 40-50 wherein, optionally, the host cell is a T cell, further optionally a human T cell.

59. A host cell comprising the means of any one of claims 51-54, wherein, optionally, the host cell is a T cell, further optionally a human T cell.

60. The host cell of any one of claims 55-59, wherein the modified cell comprises a hematopoietic progenitor cell and / or a human immune cell and / or a stem cell, optionally a pluripotent stem cell, further optionally an induced pluripotent stem cell.

61. The host cell of claim 60, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.

62. The host cell of claim 61, wherein the immune cell comprises a CD4+T cell, a CD8+T cell, a CD4' CD8' double negative T cell, a y5 T cell, a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4+T cell and a CD8+T cell, wherein, further optionally, the CD4+T cell, the CD8+T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 coreceptor a chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor P chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor P chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

63. The host cell of any one of claims 55-62, wherein the modified cell or host cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4gene; an HL A component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.

64. The host cell of claim 63, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an a2 macroglobulin gene, an a3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.

65. The host cell of claim 63 or 64, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.

66. A composition comprising the host cell of any one of claims 55-65 and a pharmaceutically acceptable carrier, diluent, or excipient.

67. The composition of claim 66, comprising at least about 30% modified CD4+T cells, combined with (ii) a composition comprising at least about 30% modified CD8+T cells, in about a 1 : 1 ratio.

68. The composition of claim 66, comprising a plurality of CD8+ T cells.

69. The composition of claim 66 or 67, comprising a plurality of CD4+ T cells.

70. The composition of any one of claims 66-69, wherein the composition contains substantially no naive T cells.

71. A composition comprising:(i) the binding protein of any one of claims 1-26;(ii) the polynucleotide of any one of claims 27-39;(ii) the expression vector of any one of claims 40-50;(iv) the means of any one of claims 51-54; and / or(iv) the host cell of any one of claims 55-65, and a pharmaceutically acceptable carrier, excipient, or diluent.

72. A method for treating a disease or disorder associated with a KRAS G12V mutation or a NRAS G12V mutation or a HRAS G12V mutation in a subject, the method comprising administering to the subject an effective amount of:(i) the binding protein of any one of claims 1-26;(ii) the polynucleotide of any one of claims 27-39;(iii) the expression vector of any one of claims 40-50;(iv) the means of any one of claims 51-54;(v) the host cell of any one of claims 55-65, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and wherein, optionally, the host cell is autologous, allogeneic, or syngeneic to the subject; and / or(vi) the composition of any one of claims 66-71, wherein, optionally, the method further comprises administering to the subject, or the subject has received, an effective amount of an agent that inhibits expression or activity of SPT6, wherein, further optionally, the an agent that inhibits expression or activity of SPT6 comprises an siRNA, e.g., as available from Creative Biolabs (e.g., at creative-biolabs.com / gene- therapy / small-interfering-ma-sirna-anti-human-supt6h-42027.htm) or from Origene (e.g.. at origene.com / catalog / rnai / sirna-oligo-duplexes / sr304661 / spt6-supt6h-human-sima-oligo-duplex- locus-id-6830).

73. The method of claim 72, wherein the disease or disorder comprises cells that express HL A- A* 02:01.

74. The method of claim 73, wherein the disease or disorder comprises cells that present a peptide:HLA complex, wherein the amino acid sequence of the peptide is KLVVVGAVGV and wherein, in at least some of the cells, the N-terminal lysine of the peptide comprises a side chain bearing one, two, or three methyl groups.

75. The method of any one of claims 72-74, wherein the disease or disorder comprises a cancer, wherein the cancer is optionally a solid cancer or a hematological malignancy.

76. The method of any one of claims 72-75, wherein the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma(PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

77. The method of any one of claims 72-76, wherein the binding protein, polynucleotide, vector, host cell, means, or composition is administered to the subject parenterally or intravenously.

78. The method of any one of claims 72-77, wherein the method comprises administering a plurality of doses of any one or more of (i)-(vi) to the subject.

79. The method of claim 78, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.

80. The method of any one of claims 72-79, wherein the composition comprises the host cell or the composition comprising the host cell, and wherein the method comprisesadministering the host cell or composition to the subject at a dose of about 104cells / kg to about 1011cells / kg.

81. The method of any one of claims 72-80, wherein the method comprises administering to the subject at least 5xl0A8, at least lxlOA9, at least 5xlOA9, at least lxl0A10, at least 1.5xl0A10, at least 2xl0A10, or at least 5xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

82. The method of any one of claims 72-81, wherein the method comprises administering to the subject at most 5xlOA9, at most lxl0A10, at most 1.5xl0A10, at most 2xl0A10, at most 5xl0A10, at most lxlOAl 1, or at most 5xl0Al 1 viable host cells that comprise the binding protein, optionally in a single dose.

83. The method of any one of claims 72-81, wherein the method comprises administering to the subject about 5xlOA9, about 6xlOA9, about 7xlOA9, about 8xlOA9, about 9xlOA9, about lxl0A10, about l. lxl0A10, about 1.2xl0A10, about 1.3xl0A10, about 1.4xl0A10, about 1.5xl0A10, about 1.6xl0A10, about 1.7xl0A10, about 1.8xl0A10, about 1.9xl0A10, or about 2xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

84. The method of any one of claims 72-81, wherein the method comprises administering to the subject about 5 xlOA9 to about 1 xlOAl 1, about 5 xlOA9 to about 5 xl0A10, about 5 xlOA9 to about 2 xl0A10, about 5 xlOA9 to about 1.5 xl0A10, about 5 xlOA9 to about 1 xl0A10, about 1 xl0A10 to about 1 xlOAl l, about 1 xl0A10 to about 5 xl0A10, about 1 xl0A10 to about 2 xl0A10, or about 1 xl0A10 to about 1.5 xl0A10 viable host cells that comprise the binding protein, optionally in a single dose.

85. The method of any one of claims 72-84, further comprising determining that the subject expresses HLA-A*02:01, prior to administering the binding protein, TCR, polynucleotide, vector, means, host cell, or composition, and / or determining that the subject expresses a KRAS G12V 5-14, NRAS G12V 5-14, or HRAS G12V 5-14 peptide comprising one or more methyl groups at a N-terminal lysine of the peptide prior to administering the binding protein, TCR, polynucleotide, vector, host cell, or composition.

86. The method of any one of claims 72-85, further comprising determining that the subject expresses HLA-A*02:01, prior to administering the binding protein, TCR, polynucleotide, vector, means, host cell, or composition, and determining that the subject expresses or diseased cells of the subject express a KRAS G12V 5-14, NRAS G12V 5-14, or HRAS G12V 5-14 peptide comprising one or more methyl groups at a N-terminal lysine of the peptide prior to administering the binding protein, TCR, polynucleotide, vector, host cell, or composition.

87. The method of any one of claims 72-86, wherein the method further comprises administering a cytokine to the subject.

88. The method of claim 87, wherein the cytokine comprises IL-2, IL-15, or IL-21.

89. The method of any one of claims 72-88, wherein the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

90. The binding protein of any one of claims 1-26, the polynucleotide of any one of claims 27-39, the expression vector of any one of claims 40-50, the means of any one of claims 51-54, the host cell of any one of claims 55-65, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and / or the composition of any one of claims 66-71, for use in a method for treating a disease or disorder associated with a KRAS G12V or a NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorlydifferentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

91. The binding protein of any one of claims 1-26, the polynucleotide of any one of claims 27-39, the expression vector of any one of claims 40-50, the means of any one of claims 51-54, the host cell of any one of claims 55-65, wherein, optionally, the host cell comprises a CD8+ T cell, a CD4+ T cell, or both, and / or the composition of any one of claims 66-71, for use the manufacture of a medicament for treating a disease or disorder associated with a KRAS G12V or a NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder comprises a cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and, wherein, optionally, the disease or disorder is selected from a pancreas cancer or carcinoma, optionally a pancreatic ductal adenocarcinoma (PDAC); a colorectal cancer or carcinoma; a lung cancer, optionally a non-small-cell lung carcinoma; a biliary cancer; an endometrial cancer or carcinoma; a cervical cancer; an ovarian cancer; a bladder cancer; a liver cancer; a myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; a myelodysplastic syndrome; a lymphoma such as Non-Hodgkin lymphoma; Chronic Melyomonocytic Leukemia; Acute Lymphoblastic Leukemia (ALL); a cancer of the urinary tract; a cancer of the small intestine; a breast cancer or carcinoma; a melanoma (optionally a cutaneous melanoma, an anal melanoma, or a mucosal melanoma); a glioma; a poorly differentiated thyroid gland carcinoma; a neuroblastoma; a histiocytic and dendritic cell neoplasm; neurofibromatisis Type 1; rhabdomyosarcoma; a soft tissue sarcoma; a bladder carcinoma; a sarcoma; a glioblastoma; a squamous cell lung carcinoma; an anaplasticastrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck carcinoma; head and neck squamous cell carcinoma; hepatocellular carcinoma; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T cell lymphoma; prostate carcinoma; refractory anemia with excess blasts-2; renal cell carcinoma; rhabdoid tumor; schwannoma; secondary AML; small cell lung carcinoma; therapy-related AML; thymic carcinoma; thyroid gland follicular carcinoma; malignant thyroid gland neoplasm; thyroid gland carcinoma; thyroid gland adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; thyroid gland papillary carcinoma; or an advanced or metastatic version thereof.

92. Use of a binding protein that recognizes a methylated (mono-, di-, and / or tri-) RAS (e.g., KRAS) G12V peptide for determining whether a sample comprises the methylated peptide.

93. A method of screening a binding protein or a set or library of binding proteins to determine whether the / a binding protein binds a Ras (e.g., KRAS) G12V 5-14 peptide comprising one or more (e.g., one, two, three, or more) methyl groups at the N-terminal lysine of the peptide.

94. The method of claim 92 or 93, comprising introducing the binding protein, or a cell (e.g., a human T cell) expressing the binding protein, to the peptide (e.g., the peptide in complex with an HLA-A*02:01, optionally to a cell presenting the peptide in complex with the HLA-A*02:01).

95. A method of identifying a binding protein that binds a Ras (e.g., KRAS) G12V 5- 14 peptide comprising one or more (e.g., one, two, three, or more) methyl groups at the N- terminal lysine of the peptide, the method comprising introducing the binding protein, or a cell (e.g., a human T cell) expressing the binding protein, to the peptide (e.g., the peptide in complex with an HLA-A*02:01, optionally to a cell presenting the peptide in complex with the HLA- A*02:01), and determining whether binding occurs.

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