Binding Protein Specific for RAS Neoantigen and Use Thereof

Abstract

The present disclosure provides, for example, compositions and methods for targeting Ras antigens for treating or preventing cancer. Embodiments disclosed include binding proteins that bind to Ras antigen:HLA complexes, such as T cell receptors. The disclosed binding proteins are sensitive to antigens and can induce activation of host T cells with low concentrations of peptide antigens. In certain embodiments, the binding proteins of the present disclosure are (i) amino acid sequences derived from the human proteome and / or (ii) non-alloreactive, substantially non-alloreactive, and / or have a low risk of alloreactivity against human HLA alleles. Polynucleotides encoding such binding proteins can be introduced into host cells, such as T cells, and the cells can be used in immunotherapies for treating various cancers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 342,025, filed May 13, 2022, U.S. Provisional Patent Application No. 63 / 380,551, filed October 21, 2022, and U.S. Provisional Patent Application No. 63 / 488,758, filed March 6, 2023, the entire disclosures of which are incorporated herein by reference in their entireties.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (502WO_SeqListing.xml, size: 189 kilobytes, and created on May 11, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Ras family proteins are small GTPases involved in the transmission of signals within cells, including, for example, the transduction of cell proliferation. Exemplary RAS proteins include KRAS (also known as CK-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 inhibit negative growth signaling can lead to continuous cell proliferation. KRAS is one of the most frequently mutated proto-oncogenes in various human cancers, including melanoma, endometrial, thyroid, pancreatic, colorectal, breast, ovarian, and lung cancer, as well as some cases of myeloid leukemia, such as AML. Pharmacological inhibitors targeting KRAS G12C have been developed, but primary and adaptive resistance to these inhibitors has been reported in cancers (e.g., Awad et al. NEJM 384:2382-2393 (2021)). New therapies targeting mutant RAS proteins are needed. [Brief explanation of the drawings]

[0004] [Figure 1A] Identification of KRAS G12V-specific T cell receptors (TCRs) from the T cell repertoire of healthy human donors. (Figure 1A) (Left) Schematic diagram showing the process for identifying HLA-A11-restricted mutant KRAS (mKRAS)-specific T cell lines from donor samples, and (Right) TNFα production by CD8+ T cells expressing mKRAS-specific TCRs in the absence (left) or presence (right) of mKRAS G12V peptide. [Figure 1B] Regarding the identification of KRAS G12V-specific T cell receptors (TCRs) from the T cell repertoire of healthy human donors (Figure 1B). (Top) Schematic diagram of the process for sorting and sequencing mKRAS-reactive CD8+ T cells and (bottom) the process for engineering CD8+ T cells to heterologously express mKRAS-specific TCRs. Fifty-six mKRAS-specific TCRs (G12V-specific or G12D-specific) were isolated, and sensitivity and cytotoxicity assays were performed. [Figure 1C] Regarding the identification of KRAS G12V-specific T cell receptors (TCRs) from the T cell repertoire of healthy human donors. (FIG. 1C) In vitro enrichment fold of T cell clones with and without KRAS G12V mutant peptide. [Figure 1D] Identification of KRAS G12V-specific T cell receptors (TCRs) from the T cell repertoire of healthy human donors. (Figure 1D) Activation of TCR-transduced T cells in vitro, assessed by the percentage of T cells expressing GFP under the control of the Nur77 locus, in the presence of various concentrations of KRAS G12V mutant peptide. T cells were transduced to express TCRs as indicated in the figure legend. [Figure 1E] Regarding the identification of KRAS G12V-specific T cell receptors (TCRs) from the T cell repertoire of healthy human donors (Figure 1E), Log EC50 KRAS G12V 9-mer peptide values (representing the concentration of KRAS G12V peptide required for TCR-transduced T cells to produce their half-maximal response in Nur77-expressing TCRs). [Figure 2A] The functional avidity of TCR 11NA4 (see Table 1) is shown compared to that of TCR220_21 (V-domain amino acid sequence set forth in SEQ ID NOs: 61 (Vα) and 62 (Vβ)), and TCR "BNT" (Vα domain amino acid sequence (with signal peptide) set forth in SEQ ID NO: 60, Vβ domain amino acid sequence (with signal peptide) set forth in SEQ ID NO: 59). (Figure 2A) Percentage of TCR-transduced primary CD8+ T cells expressing CD137 at the indicated concentrations of KRAS G12V peptide. (Figure 2B) Log EC50 of the TCR for KRAS G12V peptide. (Figure 2C) Percentage of TCR-transduced primary CD8+ T cells expressing IFN-γ at the indicated concentrations of KRAS G12V peptide. [Figure 2B] The functional avidity of TCR 11NA4 (see Table 1) is shown compared to that of TCR220_21 (V-domain amino acid sequences shown in SEQ ID NOs: 61 (Vα) and 62 (Vβ)), and TCR "BNT" (Vα domain amino acid sequence (with signal peptide) shown in SEQ ID NO: 60, Vβ domain amino acid sequence (with signal peptide) shown in SEQ ID NO: 59). (Figure 2B) Log EC50 of TCR for KRAS G12V peptide, [Figure 2C] The functional avidity of TCR 11NA4 (see Table 1) is shown compared to that of TCR220_21 (V-domain amino acid sequences shown in SEQ ID NOs: 61 (Vα) and 62 (Vβ)), and TCR "BNT" (Vα domain amino acid sequence (with signal peptide) shown in SEQ ID NO: 60, Vβ domain amino acid sequence (with signal peptide) shown in SEQ ID NO: 59). (Figure 2C) Percentage of TCR-transduced primary CD8+ T cells expressing IFN-γ at the indicated concentrations of KRAS G12V peptide. [Figure 2D]Figure 2 shows the functional avidity of TCR 11N4A (assessed as CD137 expression by host T cells). Figure 2D shows that TCR 11N4A-transduced T cells recognize both KRAS G12V 9-mer and 10-mer peptides. Figure 2E shows the log EC50 of the TCR for KRAS G12V 9-mer (left) and 10-mer (right) peptides, with values referring to the curves in Figure 2D, demonstrating strong functional avidity (low picomolar EC50) for TCR 11N4A. Figure 2F shows the absence of recognition of KRAS G12G wild-type peptide by TCR 11N4A. In Figure 2E, the y-axis values of the graph (from bottom to top) are -11, -10, -9, -8, -7, -6, and -5. In Figure 2F, the y-axis values of the graph (bottom to top) are 0, 20, 40, 60, 80, 100, and the x-axis text (left to right) is: no peptide, G12V7-18 10mer, G12V8-18 9mer, G12G7-18WT, G12G8-18WT. [Figure 2E] Figure 2 shows the functional avidity of TCR 11N4A (assessed as CD137 expression by host T cells). Figure 2D shows that TCR 11N4A-transduced T cells recognize both KRAS G12V 9-mer and 10-mer peptides. Figure 2E shows the log EC50 of the TCR for KRAS G12V 9-mer (left) and 10-mer (right) peptides, with values referring to the curves in Figure 2D, demonstrating strong functional avidity (low picomolar EC50) for TCR 11N4A. Figure 2F shows the absence of recognition of KRAS G12G wild-type peptide by TCR 11N4A. In Figure 2E, the y-axis values of the graph (from bottom to top) are -11, -10, -9, -8, -7, -6, and -5. In Figure 2F, the y-axis values of the graph (bottom to top) are 0, 20, 40, 60, 80, 100, and the x-axis text (left to right) is: no peptide, G12V7-18 10mer, G12V8-18 9mer, G12G7-18WT, G12G8-18WT. [Figure 2F]Figure 2 shows the functional avidity of TCR 11N4A (assessed as CD137 expression by host T cells). Figure 2D shows that TCR 11N4A-transduced T cells recognize both KRAS G12V 9-mer and 10-mer peptides. Figure 2E shows the log EC50 of the TCR for KRAS G12V 9-mer (left) and 10-mer (right) peptides, with values referring to the curves in Figure 2D, demonstrating strong functional avidity (low picomolar EC50) for TCR 11N4A. Figure 2F shows the absence of recognition of KRAS G12G wild-type peptide by TCR 11N4A. In Figure 2E, the y-axis values of the graph (from bottom to top) are -11, -10, -9, -8, -7, -6, and -5. In Figure 2F, the y-axis values of the graph (bottom to top) are 0, 20, 40, 60, 80, 100, and the x-axis text (left to right) is: no peptide, G12V7-18 10mer, G12V8-18 9mer, G12G7-18WT, G12G8-18WT. [Figure 2G] We show that transduced T cells expressing TCR 11N4A are specifically activated in response to mutant KRAS G12V peptide, but not to wild-type KRAS G12G 9mer or 10mer peptides. Sorted, purified populations of donor T cells expressing TCR 11N4A were exposed to 1 μg / mL of mutant KRAS G12V 9mer, wild-type KRAS G12G 9mer or 10mer, or no peptide control for 16 hours, and T cell activation was assessed by CD137 (4-1BB) expression. [Figure 3A] Figure 1 shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is a non-transduced negative control. [Figure 3B] Figure 1 shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is a non-transduced negative control. [Figure 3C]Figure 3C shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is an untransduced negative control. Figure 3D shows the activation of untransduced T cells or 11N4A-TCR and CD8αβ co-receptor engineered T cells by endogenous KRAS G12V presentation across various tumor cell lines. [Figure 3D] Figure 3 shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is an untransduced negative control. In Figures 3D-3F, 11N4A-TCR T cells are designated as "FH-KRAS-TCR." Figure 3D shows that 11N4A-TCR cells are endogenously processed and activated by presenting the KRAS G12V antigen across a diverse panel of tumor cell lines. (Left) Description of the tumor cell lines used in this study. (Right) The indicated tumor cell lines expressing HLA-A*11:01 and KRAS G12V were cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) at a 1:1 effector-to-target ratio for 20 hours. T cell activation was measured by CD137 surface staining by flow cytometry. [Figure 3E]Figure 3 shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is an untransduced negative control. In Figures 3D-3F, 11N4A-TCR T cells are shown as "FH-KRAS-TCR." Figure 3E shows that 11N4A-TCR cells secrete effector cytokines in response to endogenously processed and presented KRAS G12V antigen across a diverse panel of tumor cell lines. The indicated tumor cell lines expressing HLA-A*11:01 and KRAS G12V were cultured for 20 hours with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2). Supernatants were collected from the coculture activation assay shown in Figure 3D, and IFNγ, TNFα, and IL-2 cytokine secretion was analyzed by ELISA. IU = International Units calculated based on a standard curve of IFNγ recombinant protein. [Figure 3F] Figure 3 shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is an untransduced negative control. In Figures 3D-3F, 11N4A-TCR T cells are designated as "FH-KRAS-TCR." Figure 3F shows that 11N4A-TCR T cells proliferate in response to endogenously processed and presented KRAS G12V antigen across a diverse panel of tumor cell lines. The indicated tumor cell lines expressing HLA-A*11:01 and KRAS G12V were cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) at a 1:1 effector-to-target ratio for 6 days. T cell proliferation was measured by lymphocyte counting by flow cytometry. T cell counts are plotted as lymphocyte counts / μL. [Figure 3G]Figure 3G shows the recognition of HLA-A11+KRAS G12V-expressing tumor cell lines by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" is an untransduced negative control. Figure 3G shows that the diverse panel of tumor cell lines tested exhibits a range of KRAS G12V antigen expression. (Left) Western blot analysis of the indicated tumor cell lines. Cell lysates were prepared from tumor cells normalized by cell number. A KRASG12V-specific antibody (MA5-42375, Invitrogen) was used, and a GAPDH-specific antibody (AB9483, Abcam) was used as a loading control. (Right) Densitometric analysis of the Western blot data on the left quantifies the ratio of KRAS G12V expression to GAPDH expression for all tumor cell lines tested. [Figure 4A] Specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing KRAS G12V-specific TCRs in an Incuyte killing assay. In this assay, red regions of interest indicate the presence of tumor cells. (Figure 4A) Schematic of mKRAS tumor cell growth in the absence of mKRAS-specific T cells. [Figure 4B] Figure 4B shows specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing a KRAS G12V-specific TCR in an IncuCyte killing assay. In this assay, red regions indicate the presence of tumor cells. (Figure 4B) mKRAS+ / HLA-A11+ tumor cell growth curve in an IncuCyte® killing assay. Conditions tested were tumor cells alone, tumor cells + T cells transduced to express TCR 11N4A, and tumor cells transduced to express TCR220_21 as a control. The red regions on the y-axis indicate tumor cell growth. Additional tumor cells were added at 72 hours. [Figure 4C]Specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing a KRAS G12V-specific TCR in an Incuyte killing assay. In this assay, red regions of interest indicate the presence of tumor cells. (Figure 4C) Data from a separate killing assay experiment. T cells and SW480 tumor cell lines were present at the indicated effector:target ratios. [Figure 4D] Figure 4D shows the specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing a KRAS G12V-specific TCR in an Incuyte killing assay. In this assay, red target areas indicate the presence of tumor cells. (Figure 4D) 11N4A-transduced primary CD4+ and CD8+ T cells are cytotoxic against SW527, SW620, and CFPAC-1 tumor cell lines across multiple tumor cell challenges. Growth kinetics of the indicated HLA-A*11:01+, KRAS G12V-expressing tumor cell lines in a live tumor visualization assay in the presence of 11N4A-transduced (lower curve in each graph) or non-transduced (upper curve in each graph) primary T cells. Tumor cells expressing red fluorescent protein (SW527 and SW620 tumor cells) or green fluorescent protein (CFPAC-1 tumor cells) were cultured with TCR-transduced or non-transduced T cells at an effector-to-target ratio of 10:1 for approximately 145 hours, and tumor confluence is reported as a measure of tumor cell growth / viability throughout the study, as indicated. Additional tumor cells were added at approximately 50 and 90 hours. [Figure 4E]Specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing KRAS G12V-specific TCR in an Incuyte killing assay. In this assay, red target areas indicate the presence of tumor cells. (Figure 4E)–(Figure 4G): 11N4A-TCR cells are cytotoxic to a diverse panel of tumor cell lines. In a live tumor visualization assay, growth kinetics of various HLA-A*11:01+, KRAS G12V-expressing tumor cell lines cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) were shown. Red fluorescent protein-expressing tumor cells were cultured alone or with 11N4A-TCR T cells at an effector:target ratio of 10:1 for 96 hours. Tumor cell confluence, as measured by the total red target area (labeled as tumor cell confluence), was reported as a measure of tumor cell growth / viability throughout the study, as indicated. In (E)-(G), 11N4A-TCR T cells are indicated as "FH-KRAS-TCR." [Figure 4F] Specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing KRAS G12V-specific TCR in an Incuyte killing assay. In this assay, red target areas indicate the presence of tumor cells. (Figure 4E)–(Figure 4G): 11N4A-TCR cells are cytotoxic to a diverse panel of tumor cell lines. In a live tumor visualization assay, growth kinetics of various HLA-A*11:01+, KRAS G12V-expressing tumor cell lines cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) were shown. Red fluorescent protein-expressing tumor cells were cultured alone or with 11N4A-TCR T cells at an effector:target ratio of 10:1 for 96 hours. Tumor cell confluence, as measured by the total red target area (labeled as tumor cell confluence), was reported as a measure of tumor cell growth / viability throughout the study, as indicated. In (E)-(G), 11N4A-TCR T cells are indicated as "FH-KRAS-TCR." [Figure 4G]Specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing KRAS G12V-specific TCR in an Incuyte killing assay. In this assay, red target areas indicate the presence of tumor cells. (Figure 4E)–(Figure 4G): 11N4A-TCR cells are cytotoxic to a diverse panel of tumor cell lines. In a live tumor visualization assay, growth kinetics of various HLA-A*11:01+, KRAS G12V-expressing tumor cell lines cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) were shown. Red fluorescent protein-expressing tumor cells were cultured alone or with 11N4A-TCR T cells at an effector:target ratio of 10:1 for 96 hours. Tumor cell confluence, as measured by the total red target area (labeled as tumor cell confluence), was reported as a measure of tumor cell growth / viability throughout the study, as indicated. In (E)-(G), 11N4A-TCR T cells are indicated as "FH-KRAS-TCR." [Figure 5A] To characterize the peptide-binding motif of TCR 11N4A, we performed mutagenesis scanning experiments using KRAS G12 9-mer and 10-mer peptides. (FIG. 5A) Percentage of TCR-transduced T cells expressing Nur77-GFP in the presence of the G12V peptide (shown as "G12V WT") or variants of the G12V peptide, where amino acids at the indicated positions were replaced with alanine, glycine, or threonine, as shown. Top: Results of mutation scanning for the KRAS G12 9-mer peptide. Bottom: Results of mutation scanning for the KRAS G12 10-mer peptide. [Figure 5B] Mutagenesis scanning experiments using KRAS G12 9-mer and 10-mer peptides to characterize the peptide-binding motif of TCR 11N4A. (FIG. 5B) Percentage of 11N4A-transduced CD8+ T cells expressing the activation marker Nur77 (linked to a reporter gene) in the presence of the indicated 9-mer peptides. [Figure 5C]Mutagenesis scanning experiments using KRAS G12 9-mer and 10-mer peptides to characterize the peptide-binding motif of TCR 11N4A. (Figure 5C) Schematic of the workflow and results from the workflow to identify sequences from the human proteome containing sequences similar to the TCR 11N4A binding motif. [Figure 5D] Mutagenesis scanning experiments using KRAS G12 9-mer and 10-mer peptides to characterize the peptide-binding motif of TCR 11N4A. (Figure 5D) Results of searching the human proteome using the workflow shown in Figure 5C. Peptides from the human proteome were scored for predicted binding to HLA-A11. [Figure 6A] TCR 11N4A has a low risk of autoreactivity in humans (Figures 6A and 6B). Reactivity of 11N4A-transduced T cells to a panel of potentially cross-reactive peptides (see Figure 5B). [Figure 6B] TCR 11N4A has a low risk of autoreactivity in humans (Figures 6A and 6B). Reactivity of 11N4A-transduced T cells to a panel of potentially cross-reactive peptides (see Figure 5B). [Figure 6C] This indicates that TCR 11N4A has a low risk of autoreactivity in humans. (FIG. 6C) Peptide dose-response curves and (FIG. 6D) negative log EC50s of 11N4A-transduced T cells to the RAB7B peptide were calculated compared to the cognate KRAS G12V peptide. [Figure 6D] This indicates that TCR 11N4A has a low risk of autoreactivity in humans. (FIG. 6C) Peptide dose-response curves and (FIG. 6D) negative log EC50s of 11N4A-transduced T cells to the RAB7B peptide were calculated compared to the cognate KRAS G12V peptide. [Figure 6E]This indicates that TCR 11N4A has a low risk of autoreactivity in humans. (Figure 6E) Percentage of 11N4A-transduced CD8+ T cells expressing CD137 in response to overnight culture with a comprehensive panel of position-scanning peptides containing all possible amino acid substitutions at each position of the cognate KRAS G12V peptide (172 peptides). Peptides that elicited a response of more than 15% were considered positive in this assay. [Figure 6F] TCR 11N4A poses a low risk of autoreactivity in humans. (Figure 6F) (Left) Potentially cross-reactive peptides identified from a ScanProsite search for potential cross-reactive motifs identified from the data in (Figure 6E). (Right) CD137 expression (determined by flow cytometry) by sorted, purified primary CD8+ T cells transduced to express TCR 11N4A or TCR 11N4A+CD8αβ and cultured overnight with 100 ng / ml of potentially cross-reactive peptide. [Figure 6G] This shows that TCR 11N4A has a low risk of autoreactivity in humans. (Figure 6G) TCR 11N4A does not elicit cross-reactive peptide responses in vitro. T cell activation assay using 11N4A-TCR peptide-stimulated T cells generated from PBMCs of two healthy donors. TCR 11N4A-T cells were incubated with 1 μg / ml of each indicated peptide for 18 hours, followed by flow cytometry analysis of CD137 expression as a measure of T cell activation. (Left) Human self-peptides identified from alanine scanning motifs, and (right) Human self-peptides identified from XScan scanning motifs (see Table 2). [Figure 7A] Regarding screening to assess the potential alloreactivity of TCR 11N4A (Figure 7A), B-lymphoblastoid cell lines (B-LCLs) expressing different HLA alleles were incubated with 11N4A-transduced CD8+ T cells, and T cells were assessed for reactivity as determined by expression of IFN-γ or CD137. [Figure 7B]Regarding screening to assess the potential alloreactivity of TCR 11N4A. (FIG. 7B) Results from the alloreactivity screen: Percentage of CD137+ 11N4A-transduced T cells with (top) or without (bottom) CD8αβ against B-LCL expressing common HLA alleles. [Figure 8] 1 shows the killing activity of CD8+ and CD4+ T cells engineered to express TCR 11N4A and CD8αβ co-receptors against mKRAS:HLA-A11+ tumor cells. [Figure 9A] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9B] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9C] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9D] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9E] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9F] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9G] The nucleotide sequence for TCR 11N4A and expression constructs encoding or containing it are shown. [Figure 9H] 1 shows the amino acid sequence for TCR 11N4A and expression constructs encoding or containing it. [Figure 9I] 1 shows the amino acid sequence for TCR 11N4A and expression constructs encoding or containing it. [Figure 9J] 1 shows the amino acid sequence for TCR 11N4A and expression constructs encoding or containing it. [Figure 10A]The nucleotide sequence for TCR 11N6 and expression constructs encoding or containing it are shown. [Figure 10B] The nucleotide sequence for TCR 11N6 and expression constructs encoding or containing it are shown. [Figure 10C] The nucleotide sequence for TCR 11N6 and expression constructs encoding or containing it are shown. [Figure 10D] The nucleotide sequence for TCR 11N6 and expression constructs encoding or containing it are shown. [Figure 10E] The nucleotide sequence for TCR 11N6 and expression constructs encoding or containing it are shown. [Figure 10F] 1 shows the amino acid sequence for TCR 11N6 and expression constructs encoding or containing it. [Figure 10G] 1 shows the amino acid sequence for TCR 11N6 and expression constructs encoding or containing it. [Figure 10H]

[0033] Figures 9A-10H show the amino acid sequence for TCR 11N6 and expression constructs encoding or containing it. It will be understood that not all sequences shown in Figures 9A-10H contain or are annotated with all sequence features shown in the legend. CDR3 sequences are shown according to the IMGT junction definition. [Figure 11A] Figure 11 shows the cytotoxicity of primary 11N4A TCR and CD8αβ co-receptor engineered T cells against various tumor cell lines, including SW527 (Figure 11A), CFPAC (Figure 11B), SW480 (Figure 11C), and SW620 (Figure 11D), in a repeated tumor challenge assay. T cells ("E") and target cells ("T") were at the indicated E:T ratio. [Figure 11B]Figure 11 shows the cytotoxicity of primary 11N4A TCR and CD8αβ co-receptor engineered T cells against various tumor cell lines, including SW527 (Figure 11A), CFPAC (Figure 11B), SW480 (Figure 11C), and SW620 (Figure 11D), in a repeated tumor challenge assay. T cells ("E") and target cells ("T") were at the indicated E:T ratio. [Figure 11C] Figure 11 shows the cytotoxicity of primary 11N4A TCR and CD8αβ co-receptor engineered T cells against various tumor cell lines, including SW527 (Figure 11A), CFPAC (Figure 11B), SW480 (Figure 11C), and SW620 (Figure 11D), in a repeated tumor challenge assay. T cells ("E") and target cells ("T") were at the indicated E:T ratio. [Figure 11D] Figure 11 shows the cytotoxicity of primary 11N4A TCR and CD8αβ co-receptor engineered T cells against various tumor cell lines, including SW527 (Figure 11A), CFPAC (Figure 11B), SW480 (Figure 11C), and SW620 (Figure 11D), in a repeated tumor challenge assay. T cells ("E") and target cells ("T") were at the indicated E:T ratio. [Figure 12A] Figure 12 shows the robust in vivo antitumor activity of primary CD4+ and CD8+ T cells engineered to express the 11N4A TCR and CD8αβ co-receptor in SW527 (Figure 12A), CFPAC (Figure 12B), and SW620 (Figure 12C) tumor challenge models. [Figure 12B] Figure 12 shows the robust in vivo antitumor activity of primary CD4+ and CD8+ T cells engineered to express the 11N4A TCR and CD8αβ co-receptor in SW527 (Figure 12A), CFPAC (Figure 12B), and SW620 (Figure 12C) tumor challenge models. [Figure 12C] Figure 12 shows the robust in vivo antitumor activity of primary CD4+ and CD8+ T cells engineered to express the 11N4A TCR and CD8αβ co-receptor in SW527 (Figure 12A), CFPAC (Figure 12B), and SW620 (Figure 12C) tumor challenge models. [Figure 13A]Combination treatment with 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells and 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells demonstrates improved in vitro and in vivo anti-tumor efficacy compared with monotherapy with 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells or 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells (Figure 13A). Growth kinetics of the HLA-A11+ KRAS G12V-expressing CFPAC-1 tumor cell line was measured in the presence of 11N4A TCR-CD8αβ co-receptor-engineered CD4+ T cells, 11N4A TCR-CD8αβ co-receptor-engineered CD8+ T cells, or 11N4A TCR-CD8αβ co-receptor-engineered CD4+ and CD8+ T cells. [Figure 13B] Combination treatment of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells and 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells demonstrates improved in vitro and in vivo anti-tumor efficacy compared with monotherapy of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells or 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells (Figure 13B). Tumor kinetics of CFPAC-Luc tumor cells inoculated into NSG immunodeficient mice was measured after intraperitoneal treatment with untransduced CD4+ and CD8+ T cells, 11N4A TCR-CD8αβ co-receptor-engineered CD4+ T cells, 11N4A TCR-CD8αβ co-receptor-engineered CD8+ T cells, or 11N4A TCR-CD8αβ co-receptor-engineered CD4+ and CD8+ T cells, respectively. [Figure 13C]Combination treatment of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells and 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells demonstrates improved in vitro and in vivo anti-tumor efficacy compared to monotherapy of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells or 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells (Figures 13C-E). Combining a CD8αβ co-receptor (also referred to as a "CD8α / β" co-receptor) with a class I TCR improves the anti-tumor response of TCR-engineered T cells. Growth kinetics of an HLA-A*11:01+, KRAS G12V-expressing tumor cell line (OVCAR-5) in the presence of primary CD4+ T cells alone, CD8+ T cells alone, or a combination (1:1 ratio) of CD4+ / CD8+ T cells transduced with TCR 11N4A (Figure 13C) or TCR 11N4A+CD8αβ co-receptor (Figure 13D). A negative control tumor cell line (PANC1) that does not express KRAS G12V was used (Figure 13E). Red fluorescent protein-expressing tumor cells were cultured alone or with TCR-transduced T cells at a 1:1 effector-to-target ratio for 172 hours, and tumor cell confluence, measured by NucLight Red total red target area, was reported throughout the study as a measure of tumor cell growth / viability, as indicated. Additional tumor cells were added at 72 and 108 hours. [Figure 13D]Combination treatment of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells and 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells demonstrates improved in vitro and in vivo anti-tumor efficacy compared to monotherapy of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells or 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells (Figures 13C-E). Combining a CD8αβ co-receptor (also referred to as a "CD8α / β" co-receptor) with a class I TCR improves the anti-tumor response of TCR-engineered T cells. Growth kinetics of an HLA-A*11:01+, KRAS G12V-expressing tumor cell line (OVCAR-5) in the presence of primary CD4+ T cells alone, CD8+ T cells alone, or a combination (1:1 ratio) of CD4+ / CD8+ T cells transduced with TCR 11N4A (Figure 13C) or TCR 11N4A+CD8αβ co-receptor (Figure 13D). A negative control tumor cell line (PANC1) that does not express KRAS G12V was used (Figure 13E). Red fluorescent protein-expressing tumor cells were cultured alone or with TCR-transduced T cells at a 1:1 effector-to-target ratio for 172 hours, and tumor cell confluence, measured by NucLight Red total red target area, was reported throughout the study as a measure of tumor cell growth / viability, as indicated. Additional tumor cells were added at 72 and 108 hours. [Figure 13E]Combination treatment of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells and 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells demonstrates improved in vitro and in vivo anti-tumor efficacy compared to monotherapy of 11N4A TCR and CD8αβ co-receptor-engineered CD4+ T cells or 11N4A TCR and CD8αβ co-receptor-engineered CD8+ T cells (Figures 13C-E). Combining a CD8αβ co-receptor (also referred to as a "CD8α / β" co-receptor) with a class I TCR improves the anti-tumor response of TCR-engineered T cells. Growth kinetics of an HLA-A*11:01+, KRAS G12V-expressing tumor cell line (OVCAR-5) in the presence of primary CD4+ T cells alone, CD8+ T cells alone, or a combination (1:1 ratio) of CD4+ / CD8+ T cells transduced with TCR 11N4A (Figure 13C) or TCR 11N4A+CD8αβ co-receptor (Figure 13D). A negative control tumor cell line (PANC1) that does not express KRAS G12V was used (Figure 13E). Red fluorescent protein-expressing tumor cells were cultured alone or with TCR-transduced T cells at a 1:1 effector-to-target ratio for 172 hours, and tumor cell confluence, measured by NucLight Red total red target area, was reported throughout the study as a measure of tumor cell growth / viability, as indicated. Additional tumor cells were added at 72 and 108 hours. [Figure 14] Figure 1 shows that T cells transduced with TCR 11N4A and CD8αβ co-receptor did not exhibit cytokine-independent growth in vitro. [Figure 15] The lack of response of the 11N4A-TCR mimetic T cell product to the RAB7B peptide is shown. The KRAS G12V index peptide (positive control) and RAB7B peptide doses ranging from 10 to 0.00001 mg / mL were tested to evaluate the reactivity of the 11N4A-TCR mimetic T cell product generated from two donors. Titrating doses of 9-mer and 10-mer KRAS G12V and RAB7B peptides were exogenously added to 11N4A-TCR T cells for 16 hours, followed by flow cytometry analysis of CD137 expression by the T cells. [Figure 16] This figure shows the lack of response of 11N4A-TCR mimic T cell products to overexpressed, endogenously processed, and presented RAB7B. HEK293 cells or HeLa cells expressing standard (SP) or immunoproteasome (IP) subunits were engineered to express HLA-A11 and full-length RAB7B proteins. CFPAC-1 (KRAS G12V+) and PANC1 (KRAS G12V-) were used as positive and negative controls, respectively. Sorted CD4+ or CD8+ 11N4A-TCR or untransduced (UTD) T cell products were co-cultured with each cell line for 16 hours, followed by flow cytometry analysis of CD137 expression by T cells. In this figure, 11N4A-TCR T cells are designated as "FH-KRAS-TCR." [Figure 17] This shows that the 11N4A-TCR mimetic product does not exhibit cell growth in the absence of cytokines. The 11N4A-TCR T cell product was enriched for KRAS-G12V A11-tetramer-positive T cells and expanded in two donors in vivo for 10 days in 15+5% serum replacement medium + 100 U / mL IL-2 using anti-CD3 and anti-CD28 beads. Untransduced primary T cells (UTD) from the same donors were expanded in parallel for 10 days. On day 10, the cells were washed and resuspended in 15+5% serum medium in vivo lacking cytokines, and cell growth kinetics was measured over a 35-day period. In Figure 17, the 11N4A-TCR T cells are designated as "FH-KRAS-TCR." DETAILED DESCRIPTION OF THE INVENTION

[0005] The present disclosure generally relates to binding proteins specific for Ras neoantigens, modified host (e.g., immune) cells expressing the same, polynucleotides encoding the binding proteins, and related uses. Mutated Ras proteins (e.g., KRAS, NRAS, HRAS) can produce neoantigens that contain 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: 78; Uniprot KB P01111), or at position 12 of the full-length HRAS protein (SEQ ID NO: 79; Uniprot KB P01112).

[0006] The present disclosure provides binding proteins capable of binding to Ras neoantigens. In certain aspects, binding proteins comprising a TCR Vα domain and a TCR Vβ domain (and a host cell, e.g., an immune cell, comprising a heterologous polynucleotide encoding a Ras-specific binding protein of the present disclosure) are provided, which are capable of binding to a Ras peptide antigen:HLA complex, wherein the Ras peptide antigen comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 2 or 3. In certain embodiments, the HLA comprises HLA-A*11, such as HLA-A*11:01.

[0007] The disclosed binding proteins are highly sensitive to antigens and, in certain embodiments, can induce host T cell activation at low concentrations of peptide antigen. In certain embodiments of a population or sample of (e.g., CD8+ or CD4+) T cells expressing the binding proteins, the T cells have half-maximal expression of the activation marker Nur77 in the presence of the peptide at a LogEC50 of less than -9 M (e.g., between -9 M and -10 M). In certain embodiments of a population or sample of (e.g., CD8+ or CD4+) T cells expressing the binding proteins, the T cells have half-maximal expression of CD137 in the presence of a LogEC50 of less than -10 M (e.g., between -10 M and -11 M). In certain embodiments of a population or sample of (e.g., CD8+ or CD4+) T cells expressing the binding proteins, the T cells have half-maximal expression of IFN-γ in the presence of a peptide at a LogEC50 of less than -10 M (e.g., between -10 M and -11 M).

[0008] Host (e.g., T) cells expressing binding proteins according to the present disclosure are activated (e.g., as determined by expression of CD137) in the presence of mutant KRAS-expressing cancer cell lines, including OVCAR5 (ovarian serous adenocarcinoma), DAN-G (pancreatic adenocarcinoma), CFPAC1 (pancreatic adenocarcinoma), SW480 (colon cancer), SW527 (breast cancer), and NCI-H441 (lung adenocarcinoma) cell lines.

[0009] In some embodiments, host cells (e.g., T cells, such as CD4+ T cells or CD8+ T cells) expressing a binding protein according to the present disclosure can specifically kill mutant KRAS-expressing cells (e.g., SW480 cells at an 8:1 effector:target ratio, a 4:1 effector:target ratio, or a 2:1 effector:target ratio, etc.) for more than 144 hours in vitro, including when additional tumor cells (i.e., additional mutant KRAS-expressing cells) are added at 72 hours in a re-challenge setting.

[0010] In certain embodiments, the binding proteins of the disclosure are non-alloreactive, substantially non-alloreactive, and / or have a low risk of alloreactivity with (i) amino acid sequences derived from the human proteome, and / or (ii) human HLA alleles.

[0011] In any of the embodiments disclosed herein, the binding proteins can be human, humanized, or chimeric. Also provided are polynucleotides encoding the binding proteins, vectors containing the polynucleotides, and host cells containing the polynucleotides and / or vectors and / or expressing the binding proteins. The binding proteins and host cells (e.g., T cells, NK cells, NK-T cells) disclosed herein are useful for treating diseases or disorders associated with KRAS neoantigens, such as cancer. The binding proteins disclosed herein can also bind to the G12V antigen that occurs in human NRAS or human HRAS, which protein contains sequence identical to KRAS in a region near residue G12. Thus, the disclosed compositions are useful for treating diseases or disorders associated with KRAS neoantigens containing a G12V mutation, NRAS neoantigens containing a G12V mutation, or HRAS neoantigens containing a G12V mutation, or any combination thereof.

[0012] Also provided are methods and uses of the binding proteins, polynucleotides, vectors, host cells, and related compositions disclosed herein for the treatment of diseases or disorders associated with KRAS, NRAS, and / or HRAS mutations as provided herein.

[0013] Also provided are methods comprising introducing a polynucleotide encoding a binding protein of the present disclosure (or introducing a vector comprising the polynucleotide) into a host cell, or into a population or sample of a plurality of host cells. In some embodiments, the polynucleotide or vector further encodes a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain, a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain, or both. In certain embodiments, the host cell(s) comprise T cells, such as CD4+ T cells or CD8+ T cells. In certain embodiments, the host cell(s) comprise primary T cells. In certain embodiments, the host cell(s) comprise peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises culturing the host cell(s). In some embodiments, the host cell(s) are derived from a subject with a disease or disorder associated with a KRAS G12V or NRAS G12V or HRAS G12V mutation. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the subject is positive for expression of HLA-A11, such as HLA-A*11:01. In certain embodiments, the host cell(s) are from a healthy subject. In some embodiments, the method is performed in vitro. In other embodiments, the method is performed ex vivo. Also provided are host cells, host cell populations, or host cell samples produced by the methods. In some embodiments, the host cell population comprises CD8+ T cells, CD4+ T cells, or both. In some embodiments, the method further comprises selecting and combining CD8+ T cells with CD4+ T cells to provide a composition comprising CD8+ T cells and CD4+ T cells in a ratio of about 1:1.

[0014] Before describing the present disclosure in more detail, it may be helpful to an understanding of the present disclosure to provide definitions of certain terms used herein. Additional definitions are set forth throughout the disclosure.

[0015] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the recited range, and fractions thereof (such as tenths and hundredths of integers), where appropriate, unless otherwise indicated. Also, any numerical range recited herein with respect to any physical characteristic, such as polymer subunits, size, or thickness, should be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ±20% of the recited range, value, or structure, unless otherwise indicated. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components. The use of alternatives (e.g., "or") should be understood to mean either one of the alternatives, both, or any combination thereof. As used herein, the terms "include," "having," and "comprise" are used interchangeably, and the terms and variations thereof are intended to be non-limiting.

[0016] Additionally, it should be understood that each individual compound or group of compounds derived from the various combinations of structures and substituents described herein is disclosed by this application to the same extent as if each compound or group of compounds were individually indicated. Thus, selection of a particular structure or particular substituents is within the scope of this disclosure.

[0017] The term "consisting essentially of" is not equivalent to "comprising" and refers to specified materials or steps in a claim or that do not materially affect the basic characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module), or protein (which may have one or more domains, regions, or modules) "consists essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein includes extensions, deletions, mutations, or combinations thereof (e.g., amino- or carboxy-terminal or inter-domain amino acids) 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 the domain, region, module, or protein and do not substantially affect (i.e., reduce activity by more than 50%, such as by no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein(s) (e.g., target binding affinity or avidity of a binding protein).

[0018] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Protein applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids 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.

[0019] As used herein, a "hematopoietic progenitor cell" is a hematopoietic stem cell or cell that may be derived from fetal tissue and is capable of further differentiation into a mature cell type (e.g., an immune system cell). Exemplary hematopoietic progenitor cells include those expressing CD24 Lo Lin - CD117 +These include those that have a phenotype similar to that of thymocytes or those found in the thymus (called progenitor thymocytes).

[0020] As used herein, "immune system cell" means any cell of the immune system that arises from hematopoietic stem cells in the bone marrow, which give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells, e.g., monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes), and lymphoid progenitor cells (which give rise to lymphoid cells, e.g., T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 - CD8 - These include double-negative T cells, γδ T cells, regulatory T cells, natural killer cells, natural killer T cells, and dendritic cells. Macrophages and dendritic cells can also 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 the T cell.

[0021] A "T cell" or "T lymphocyte" is a cell of the immune system that matures in the thymus and produces a T cell receptor (TCR). T cells are naive ("T N"; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased or no expression of CD45RO 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 and cytotoxic). TM can be further divided into subsets of central memory T cells (TCM expressing CD62L, CCR7, CD28, CD95, CD45RO, and CD127) and effector memory T cells (TEM expressing CD45RO and decreased expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TE) refer to antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have reduced expression of CD62L, CCR7, and CD28 compared to TCM, and are granzyme and perforin positive (e.g., upon stimulation). Helper T cells (T H ) is a CD4 receptor that affects the activity of other immune cells by releasing cytokines. + CD4 cells + T cells can activate and suppress adaptive immune responses, and which of these two functions is induced depends, for example, on the presence of transcription factors and other cells and signals. T cells can be collected using known techniques, and various subpopulations or combinations thereof can be enriched or depleted by known techniques, for example, by binding affinity to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, e.g., CD4 + CD25 + (Foxp3 + ) Regulatory T cells and Treg17 cells, as well as Tr1, Th3, and CD8 + CD28 - , and Qa-1-restricted T cells.

[0022] "T cell receptor" (TCR) refers to a member of the immunoglobulin superfamily that has 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), and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. TCRs may be found on the surface of cells or in a soluble form, and generally comprise a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively) or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). In certain embodiments, polynucleotides encoding binding proteins, e.g., TCRs, of the present disclosure can be codon-optimized to enhance expression in particular host cells, such as cells of the immune system, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, or natural killer T cells (Scholten et al., Clin. Immunol. 119:135, 2006). Exemplary T cells capable of expressing the binding proteins and TCRs of the present disclosure include CD4 + T cells, CD8 + These include T cells and their associated subpopulations (e.g., naive, central memory, stem cell memory, effector memory).

[0023] Similar to immunoglobulins (e.g., antibodies), the extracellular portions of TCR chains (e.g., α chain, β chain) can contain two immunoglobulin domains: an N-terminal variable domain (e.g., an α chain variable domain or Vα; a β chain variable domain or Vβ; typically amino acids 1-116 according to Kabat numbering (Kabat, et al., "Sequences of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)) and one constant domain adjacent to the cell membrane (e.g., an α chain constant domain or Cα, typically amino acids 117-259 according to Kabat; a β chain constant domain or Cβ, typically amino acids 117-295 according to Kabat). Also, similar to immunoglobulins, variable domains contain complementarity 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., EMBOJ. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The source of the TCRs used in the present disclosure may be derived from various animal species, such as human, mouse, rat, rabbit, or other mammals.

[0024] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR α or β chain (or γ and δ chains for γδ TCRs)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen. The variable domains of the α and β chains of naturally occurring TCRs (Vα and Vβ, respectively) generally have a similar structure, with each domain containing four generally conserved framework regions (FRs) and three CDRs. The Vα domain is encoded by two separate DNA segments, a variable gene segment and a joining gene segment (VJ); the Vβ domain is encoded by three separate DNA segments, a variable gene segment, a diversity gene segment, and a joining gene segment (VDJ). A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind to a particular antigen can be isolated using the Vα or Vβ domains from the TCR that bind the antigen to screen a library of complementary Vα or Vβ domains, respectively.

[0025] 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 an immunoglobulin (e.g., TCR) variable region. CDRs confer antigen specificity and binding affinity and are separated from each other in the primary amino acid sequence by framework regions. Generally, each TCR α chain variable region has three CDRs (αCDR1, αCDR2, αCDR3 (also identified as CDR1α, CDR2α, and CDR3α, respectively)), and each TCR β chain variable region has three CDRs (βCDR1, βCDR2, βCDR3 (also identified as CDR1β, CDR2β, and CDR3β, respectively)). In TCRs, CDR3 is considered the primary CDR responsible for recognizing processed antigen. Generally, CDR1 and CDR2 interact primarily or exclusively with MHC.

[0026] CDR1 and CDR2 are encoded within the variable gene segments of the TCR variable region coding sequence, while CDR3 is encoded by a region spanning the variable and joining segments for Vα, or the variable, diversity, and joining segments for Vβ. Thus, if the identities of the variable gene segments of Vα or Vβ are known, the sequences of their corresponding CDR1 and CDR2 can be deduced, for example, according to the numbering scheme described herein. Compared to CDR1 and CDR2, CDR3, particularly CDR3β, is typically significantly more variable due to the addition or loss of nucleotides during the recombination process.

[0027] TCR variable domain sequences can be aligned to numbering schemes (e.g., Kabat, Chothia, EU, IMGT, Extended Chothia, and Aho), which allow equivalent residue positions to be annotated and different molecules to be compared, for example, using the ANARCI software tool (2016, Bioinformatics 15:298-300). The numbering schemes provide a standard delineation of framework regions and CDRs in TCR variable domains. In certain embodiments, the CDRs of the present disclosure are identified or defined according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003, imgt.org / IMGTindex / V-QUEST.php). In some embodiments, a CDR (e.g., CDR3) is identified or defined according to the IMGT junction definition. In some embodiments, a CDR (e.g., CDR3 of a binding protein, or all six CDRs) is identified or defined according to the IMGT definition (or scheme or method). Examples of CDRs identified or defined according to IMGT are provided in SEQ ID NOs: 14-17 (with reference to Vα or TCRα of 11N4A), 24-27 (with reference to Vβ or TCRβ of TCR 11N4A), 40-43 (with reference to Vα or TCRα of TCR 11N6), and 50-53 (with reference to Vβ or TCRβ of TCR 11N6). In some embodiments, the CDRs of the disclosure (or all six CDRs of a binding protein) are identified or defined according to the Kabat numbering scheme or method. In some embodiments, the CDRs of the disclosure (or all six CDRs of a binding protein) are identified or defined according to the Chothia numbering scheme or method. In some embodiments, the CDRs of the disclosure (or all six CDRs of a binding protein) are identified or defined according to the EU numbering scheme or method. In some embodiments, the CDRs of the disclosure (or all six CDRs of a binding protein) are identified or defined according to the extended Chothia numbering scheme or method. In some embodiments, the CDRs of the disclosure (or all six CDRs of a binding protein) are identified or defined according to the Aho numbering scheme or method.

[0028] The source of the TCRs used in the present disclosure can be derived from any of a variety of animal species, such as humans, mice, rats, rabbits, or other mammals. The TCR constant domain sequences can be derived, for example, from humans, mice, marsupials (e.g., opossums, bandicoots, wallabies), sharks, or non-human primates. In certain preferred embodiments, the TCR constant domain sequences are human or comprise engineered variants of human sequences. The TCR constant domains can be engineered to improve, for example, pairing, expression, stability, or any combination thereof. See, for example, 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 Cα and Cβ include mutating native amino acids to cysteines such that a disulfide bond is formed between the introduced cysteine in one TCR constant domain and the native cysteine in the other TCR constant domain. Such mutations can include, for example, T48C in Cα, T57C or S57C in Cβ, or both. Also provided are embodiments in which the cognate TCR constant domains comprise mutations such that, for example, one TCR constant domain (e.g., one of Cα and Cβ) comprises an introduced so-called "cavity" (e.g., obtained by replacing one or more naturally occurring amino acids with one or more amino acids having smaller side chains) and the other (e.g., the other of Cα and Cβ) comprises a compensatory so-called "protrusion" (e.g., obtained by replacing one or more naturally occurring amino acids with one or more amino acids having larger side chains), resembling the "knobs-into-holes" configuration used to promote preferential pairing of antibody heavy chains. Also provided are embodiments in which TCR constant domain amino acids are mutated to introduce or modify charge properties to favor pairing of the mutated constant domain.Examples of mutations that can be made in Cα and Cβ to promote specific pairing by knobs-into-hole or charge-pairing mechanisms are provided in Voss et al., J. Immunol 180(1):391-401 (2008) doi.org / 10.4049 / jimmunol.180.1.391. See also U.S. Patent No. 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.

[0029] Mutations to improve stability may include a mutation in the Cα transmembrane domain from the sequence LSVIGF to the sequence LLVIVL ("LVL" mutation; see Haga-Friedman et al., J Immunol 188:5538-5546 (2012), the TCR mutations and mutant TCR constant domain sequences of which are incorporated herein by reference).

[0030] As used herein, the term "CD8 coreceptor" or "CD8" refers to the cell surface glycoprotein CD8 as either an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 coreceptor supports the function of cytotoxic T cells (CD8+) and functions through signal transduction via the 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 human CD8 beta chain isoforms (see UniProtKB identifier P10966) and a single human CD8 alpha chain isoform (see UniProtKB identifier P01732).

[0031] "CD4" is an immunoglobulin coreceptor 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 contains four immunoglobulin domains (D1-D4) expressed on the cell surface. During antigen presentation, CD4 is recruited along with the TCR complex and binds to different regions of the MHC II molecule (CD4 binds to MHC II β2, while the TCR complex binds to MHC II α1 / β1). Without wishing to be bound by theory, it is believed that proximity to the TCR complex allows CD4-associated kinase molecules to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present on the cytoplasmic domain of CD3. This activity is thought to amplify the signals generated by activated TCRs and the immune response to generate or recruit various types of immune system cells, including T helper cells.

[0032] In certain embodiments, TCRs are found on the surface of T cells (or T lymphocytes) and associate with the CD3 complex. "CD3" is a six-chain multiprotein complex involved in antigen signaling in T cells (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, the complex comprises a homodimer of one CD3γ chain, one CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3β, and CD3ε chains are related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3β, and CD3ε chains are negatively charged, which is thought to allow these chains to associate with positively charged regions of the T cell receptor chains. The intracellular tails of the CD3γ, CD3β, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, with each CD3ζ chain having three. Without wishing to be bound by theory, ITAMs are thought to be important for the signaling capacity of the TCR complex. The CD3 used in this disclosure may be derived from a variety of animal species, including human, mouse, rat, or other mammals.

[0033] As used herein, "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, the TCR complex may be composed of a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, the TCR complex may be composed of a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRβ chain.

[0034] "Component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a partial TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0035] "Chimeric antigen receptor" (CAR) refers to a fusion protein that is not naturally occurring or that has been engineered to contain two or more naturally occurring amino acid sequences, domains, or motifs linked together in a way that does not naturally occur in a host cell, and which fusion protein can function as a receptor when present on the surface of a cell. A CAR can comprise an antigen-binding domain (e.g., derived from or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived from or derived from a TCR specific for a cancer antigen, an scFv derived from or derived from an antibody, or an antigen-binding domain derived from or derived from a killer immune receptor from an NK cell) linked to a transmembrane domain, and an extracellular portion comprising one or more intracellular signaling domains (optionally containing costimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); also see 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), the CAR construct and methods for making the same are incorporated herein by reference. CARs of the present disclosure that specifically bind to Ras antigens (e.g., in the context of a peptide:HLA complex) comprise TCR Vα and Vβ domains.

[0036] Any polypeptide of the present disclosure, when encoded by a polynucleotide sequence, can include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to the appropriate location within or outside the cell (e.g., inserted into or localized in the cell membrane, secreted by the cell, or contained within the cell). In some contexts, the signal peptide is about 15 to about 22 amino acids in length. The signal peptide may be removed from the polypeptide during localization (e.g., membrane insertion) or secretion, or once localization (e.g., membrane insertion) or secretion is complete. In some embodiments, the signal peptide is completely removed from the polypeptide. In some embodiments, typically no more than 1, 2, 3, 4, 5, or 6 amino acids, but not all, of the signal peptide remain in the polypeptide, and the remainder of the signal peptide is removed. A polypeptide having a signal peptide is referred to herein as a "preprotein," and a polypeptide from which the signal peptide has been removed is referred to herein as a "mature" protein or polypeptide. In any of the embodiments disclosed herein, the binding protein or fusion protein comprises or is a mature protein, or is or includes a preprotein.

[0037] "Linker" refers to an amino acid sequence that can connect two proteins, polypeptides, peptides, domains, regions, or motifs and provide a spacer function compatible with the interaction of two sub-binding domains such that the resulting polypeptide retains specific binding affinity to a target molecule (e.g., scTCR) or signaling activity (e.g., TCR complex). In certain embodiments, the linker is composed of about 2 to about 35 amino acids, for example, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids. Exemplary linkers include glycine-serine linkers.

[0038] "Antigen" or "Ag," as used herein, refers to an immunogenic molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells (e.g., T cells), or both. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, or lipid. It is readily apparent that antigens may be synthetic, recombinantly produced, or derived from a biological sample. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens may be produced by cells that have been modified or genetically engineered to express the antigen or by cells that endogenously (e.g., without human modification or genetic engineering) express an immunogenic mutation or polymorphism.

[0039] "Neoantigen," as used herein, refers to a host cell product containing structural changes, modifications, or mutations that create a new antigen or antigenic epitope not previously observed in the subject's genome (i.e., in a sample of healthy tissue from the subject) or not "seen" or recognized by the host's immune system; (a) processed by the cellular antigen processing and transport machinery, presented on the cell surface in association with an MHC (e.g., HLA) molecule, and (b) eliciting an immune response (e.g., a cellular (T cell) response). Neoantigens can result, for example, from a coding polynucleotide with an alteration (substitution, addition, deletion) that results 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 an environmental agent (e.g., chemical, radiological) that results in a genetic change. Neoantigens can arise separately from tumor antigens, or can arise from or be associated with tumor antigens. "Tumor neoantigen" (or "tumor-specific neoantigen") refers to a protein containing a neoantigenic determinant associated with, arising from, or arising in a tumor cell or multiple cells within a tumor. Tumor neo-antigenic determinants are found, for example, on antigenic tumor proteins or peptides containing one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g., pancreatic cancer, lung cancer, colorectal cancer), as well as proteins or peptides derived from viral open reading frames associated with virus-associated tumors (e.g., cervical cancer, some head and neck cancers). The terms "antigen" and "neo-antigen" are used interchangeably herein when referring to Ras antigens containing the mutations disclosed herein.

[0040] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant 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.

[0041] As used herein, the term "KRAS (or NRAS or HRAS) antigen (or neo-antigen)" or "KRAS (or NRAS or HRAS) peptide antigen (or neo-antigen)" or "KRAS (NRAS or HRAS) peptide" refers to a naturally or synthetically produced peptide portion of a KRAS or NRAS or HRAS protein that ranges in length from about 7, about 8, about 9, or about 10 amino acids to about 20 amino acids and contains at least one amino acid alteration caused by a G12 (e.g., G12V) mutation (position 12 based on the full-length KRAS protein sequence set forth in SEQ ID NO: 1; also based on the full-length NRAS and HRAS protein sequences set forth in SEQ ID NOs: 78 and 79, respectively), which peptide is capable of forming a complex with an MHC (e.g., HLA) molecule, and a binding protein of the present disclosure specific for a KRAS or NRAS or HRAS peptide:MHC (e.g., HLA) complex is capable of specifically binding to such a complex. Exemplary KRAS (or NRAS or HRAS) antigens comprise, consist essentially of, or consist of a peptide having the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0042] "Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface of all nucleated cells. MHC class I molecules are heterodimers, consisting of a membrane-spanning α chain (with three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules are composed of two membrane-spanning transmembrane glycoproteins, α and β. Each chain contains two domains. MHC class I molecules deliver peptides originating from the cytosol to the cell surface, where the peptide:MHC complex is expressed by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface, where they are recognized by CD4 +They are recognized by T cells. Human MHC is called human leukocyte antigen (HLA). HLA corresponding to "class I" MHC presents peptides derived from inside cells, and examples of such HLA include HLA-A, HLA-B, and HLA-C. Alleles include HLA-A*11, such as HLA-A*11:01. HLA corresponding to "class II" MHC presents peptides derived from outside cells, and examples of such HLA include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0043] The principles of antigen presentation to T cells by antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes, or other cell types), including antigen processing by APCs and major histocompatibility complex (MHC)-restricted presentation between immunocompatible APCs (e.g., sharing at least one allelic form of an MHC gene associated with antigen presentation) and T cells, are well established (see, e.g., Murphy, Janeway's Immunobiology (2008) 133:157–160). th (Ed.) 2011 Garland Science, NY; see chapters 6, 9, and 16.) For example, processed antigenic peptides originating in the cytosol (e.g., tumor antigens, intracellular pathogens) are generally about 7 to about 11 amino acids in length and associate with class I MHC (HLA) molecules, whereas peptides processed in the vesicular system (e.g., bacteria, viruses) generally range in length from about 10 to about 25 amino acids and associate with class II MHC (HLA) molecules.

[0044] The term "KRAS-specific binding protein," as used herein, refers to a protein or polypeptide, such as, for example, a TCR, scTv, scTCR, or CAR, that binds to a KRAS or NRAS or HRAS peptide antigen (or to a KRAS or NRAS or HRAS peptide antigen:HLA complex, e.g., on a cell surface), but does not bind to peptides that do not contain the KRAS or NRAS or HRAS peptide antigen, or does not bind to HLA complexes that contain such peptides.

[0045] The binding proteins of the present disclosure, e.g., TCRs, scTvs, scTCRs, and CARs, contain target-specific binding domains. 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., a peptide, oligopeptide, polypeptide, protein) capable of specifically and non-covalently associating with, combining with, or combining with a target (e.g., a KRAS or NRAS or HRAS peptide or a KRAS or NRAS or HRAS peptide:MHC complex). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, molecular complex (i.e., a complex comprising two or more biological molecules), or other target of interest. Exemplary binding domains include immunoglobulin variable regions or single-chain constructs comprising same (e.g., single-chain TCRs (scTCRs) or scTvs).

[0046] In certain embodiments, the Ras-specific binding protein is about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K less than M dor binds to a KRAS (or NRAS or HRAS) peptide (or KRAS (or NRAS or HRAS):HLA complex) with about the same, at least about the same, or the same or greater than approximately the same affinity exhibited by exemplary Ras-specific binding proteins provided herein, such as any of the Ras-specific TCRs provided herein, e.g., as measured by the same assay. In certain embodiments, the Ras-specific binding protein comprises a Ras-specific immunoglobulin superfamily binding protein or binding portion thereof.

[0047] "Specifically binds to" or "specific for," as used herein, means that a binding protein (e.g., a TCR receptor) or binding domain (or a fusion protein thereof) binds to a target molecule, such as a TCR receptor, at a specific target site, or at a specific site, such as a TCR receptor, at a specific target site, or at a specific target ... 5 M -1 (This is the on-rate [k on ] off rate [k off ] to ] (equal to the ratio of a (i.e., the equilibrium association constant of a particular binding interaction in units 1 / M) and does not significantly associate or associate with any other molecules or components in the sample. A binding protein or binding domain (or fusion protein thereof) can be classified as a "high affinity" binding protein or binding domain (or fusion protein thereof) or a "low affinity" binding protein or binding domain (or fusion protein thereof). A "high affinity" binding protein or binding domain has an affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 K aA "low affinity" binding protein or domain refers to a binding protein or domain having a binding affinity of up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 K a Alternatively, affinity refers to the equilibrium dissociation constant (K) of a particular binding interaction in units M. d ) (e.g., 10 -5 M~10 -13 M).

[0048] In certain embodiments, a receptor or binding domain may have "enhanced affinity," which refers to a selected or engineered receptor or binding domain that has stronger binding to a target antigen than the wild-type (or parent) binding domain. For example, enhanced affinity may have a higher K for the target antigen than the wild-type binding domain. a (equilibrium association constant), K for the target antigen lower than that of the wild-type binding domain d This may be due to a lower dissociation constant (k), a lower off-rate (koff) for the target antigen than that of the wild-type binding domain, or a combination thereof.

[0049] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to particular targets and for determining the affinity of binding domains or fusion proteins, such as Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents). Binding and binding affinity can also be assessed using, for example, the fluorescence intensity observed when a binding protein binds to a labeled HLA-peptide complex or a labeled HLA-peptide complex multimer (e.g., a tetramer).

[0050] In certain embodiments, the KRAS (or NRAS, or HRAS)-specific binding domain alone (i.e., without any other portion of the KRAS (or NRAS, or HRAS)-specific binding protein) may be soluble and may be present in an amount of about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K less than M dand can bind to KRAS (or NRAS, or HRAS) (or to a KRAS (or NRAS, or HRAS) peptide, or to a KRAS (or NRAS, or HRAS) peptide:HLA complex). In certain embodiments, the KRAS (or NRAS, or HRAS)-specific binding domain comprises a KRAS (or NRAS, or HRAS)-specific scTCR (e.g., a single-chain αβTCR protein comprising Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα, or Vα-L-Vβ-Cβ, etc., where Vα and Vβ are TCR α and β variable domains, respectively, Cα and Cβ are TCR α and β constant domains, respectively, and L is a linker, such as those described herein). In some embodiments, the KRAS (or NRAS, or HRAS)-specific binding domain comprises a KRAS (or NRAS, or HRAS)-specific scTv (e.g., a single-chain TCR variable domain protein such as Vα-L-Vβ or Vβ-L-Vα, where Vα and Vβ are TCR α and β variable domains, respectively, and L is a linker such as a linker described herein).

[0051] 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 ligand concentration. Biological measures can include cytokine production (e.g., IFN-γ production, IL-2 production, etc.), cytotoxic activity, activation markers (e.g., CD137, Nur77), and proliferation. For example, T cells that respond biologically (immunologically) to a low antigen dose in vitro, such as by producing cytokines, exhibiting cytotoxic activity, or proliferating, are considered to have high functional avidity, whereas T cells with lower functional avidity require a greater amount of antigen before eliciting an immune response similar to that of high-avidity T cells. It will be understood that functional avidity differs from affinity and avidity. Affinity refers to the strength of any given binding between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to multiple antigens, in which case the overall strength of binding is the avidity.

[0052] There are numerous correlations between functional avidity and the effectiveness of immune responses. Some ex vivo studies have shown that distinct T cell functions (e.g., proliferation, cytokine 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 influence functional avidity include (a) the affinity of the TCR for the pMHC-complex, i.e., the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167:2577, 2001), (b) the expression level of the TCR, and in some embodiments, the CD4 or CD8 co-receptor, on the host cell, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), as well as T cell function and the expression level of molecules that attenuate TCR signaling.

[0053] The concentration of antigen required to induce a half-maximal response (e.g., cytokine production or activation markers by host cells; fluorescence intensity upon binding to labeled peptide:HLA multimers) between the baseline response and the maximum response after a specified exposure time is referred to as the "half-maximal effective concentration" or "EC50." EC50 values are generally expressed as molar amounts (moles / liter), but can be expressed as follows: -log 10 For example, if the EC50 is 1 μM (10 -6 M), log 10 (EC50) value is -6. Another value used is pEC50, which is the negative logarithm of the EC50 (-log 10 In the example above, an EC50 equal to 1 μM has a pEC50 value of 6. In certain embodiments, the functional avidity of a binding protein of the disclosure comprises a measure of the ability of the binding protein to promote activation and / or IFNγ production by T cells, which can be measured using assays known in the art and described herein. In certain embodiments, functional avidity comprises a measure of the ability of the binding protein to activate host cells, such as T cells, upon binding to an antigen.

[0054] The binding proteins disclosed herein can comprise high functional avidity, which can promote the induction of immune cell effector functions (e.g., activation, proliferation, cytokine production, and / or cytotoxicity) for even lower level (n e.g., HLA-A*11:01-) presented KRAS G12 mutant peptides, such as the KRAS G12V mutant peptide of SEQ ID NO:2 or SEQ ID NO:3.

[0055] In some embodiments, the binding protein has a pH of about -6.0 or less, about -6.1 or less, about -6.2 or less, about -6.3 or less, about -6.4 or less, about -6.5 or less, about -6.6 or less, about -6.7 or less, about -6.8 or less, about -6.9 or less, about -7.0 or less, about -7.1 or less, about -7.2 or less, about -7.3 or less, about -7.4 or less, about -7.5 or less, about -7.6 or less, about -7.7 or less, about -7.8 or less, about -7.9 or less. or less, about -8.0 or less, about -8.1 or less, about -8.2 or less, about -8.3 or less, about -8.4 or less, about -8.5 or less, about -8.6 or less, about -8.7 or less, about -8.8 or less, about -8.9 or less, about -9 or less, about -9.1 or less, about -9.2 or less, about -9.3 or less, about -9.4 or less, about -9.5 or less, about -9.6 or less, about -9.7 or less, about -9.8 or less, about -9.9 or less, or about -10 or less.

[0056] In some embodiments, the host cells disclosed herein have a concentration of less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, or less than about 1 pM. EC50 can be determined, for example, by assaying to identify the peptide dose that achieves half-maximal activation of a T cell population as reflected by expression of activation markers (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, Nur77, cytokines, interleukins, interferons) upon exposure to target cells in the presence of various concentrations of the variant peptide.

[0057] In some embodiments, the host cells disclosed herein have a concentration of at least about 100 mM, at least about 10 mM, at least about 1 mM, at least about 500 μM, at least about 100 μM, at least about 50 μM, at least about 10 μM, at least about 5 μM, at least about 4 μM, at least about 3 μM, at least about 2 μM, at least about 1 μM, at least about 900 nM, at least about 800 nM, at least about 700 nM, at least about 600 nM, at least about 500 nM, at least about 400 nM, at least about 300 nM, at least about 200 nM, at least about 100 nM, at least about 90 nM, at least about 80 nM, at least about 70 nM, at least about 60 nM, at least about 50 nM, at least about 40 nM, at least about 30 nM, at least about 20 nM, at least about 1 The peptide binds to the target antigen (e.g., present in the peptide:HLA (e.g., HLA-A*11:01) complex, e.g., KRAS) of the binding protein with an EC50 (e.g., a peptide dose that achieves half-maximal activation of proliferation of T cells expressing the binding protein) of 0 nM, at least about 5 nM, at least about 1 nM, at least about 900 pM, at least about 800 pM, at least about 700 pM, at least about 600 pM, at least about 500 pM, at least about 400 pM, at least about 300 pM, at least about 200 pM, at least about 100 pM, at least about 90 pM, at least about 80 pM, at least about 70 pM, at least about 60 pM, at least about 50 pM, at least about 40 pM, at least about 30 pM, at least about 20 pM, at least about 10 pM, at least about 5 pM, or at least about 1 pM. The present invention also includes a binding protein (e.g., a TCR) that binds to a KRAS G12 mutant peptide, such as a G12V mutant peptide.

[0058] In some embodiments, the binding protein (e.g., TCR) is less than about 100 mM, less than about 10 mM, less than about 1 mM, less than about 500 μM, less than about 100 μM, less than about 50 μM, less than about 10 μM, less than about 5 μM, less than about 4 μM, less than about 3 μM, less than about 2 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 or less than about 1 pM.

[0059] Fusion proteins comprising an scTCR or scTv of the present disclosure linked to a constant domain (e.g., a heavy chain constant domain or combinations thereof, e.g., Fc, CH2, CH3, CH4, and / or CH1) of an antibody (e.g., IgG(1, 2, 3, 4), IgE, IgD, IgA, IgM, and variants thereof) or fragments thereof (e.g., fragments that, in some embodiments, maintain binding to one or more Fc receptors, to C1q, to Protein A, to Protein G, or any combination thereof), and including immunoglobulin heavy chain monomers and multimers, e.g., Fc dimers, are also contemplated; see, e.g., Wong et al., J. Immunol. 198:1 Supp. (2017). For example, variant Fc polypeptides comprising mutations that enhance, reduce, or prevent binding to or by FcRn or other Fc receptors are known and are contemplated within the present disclosure.

[0060] In certain embodiments, a binding protein or fusion protein (e.g., a 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 that heterologously expresses the binding protein or fusion protein). The avidity of such host cells for a KRAS (or NRAS, or HRAS) peptide antigen or a KRAS (or NRAS, or HRAS) peptide antigen:HLA complex can be determined, for example, by 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, optionally in a peptide:HLA complex, to the host cell, and then measuring host cell activity, such as production or secretion of cytokines (e.g., IFN-γ; TNFα); increased expression of host cell signaling or activation components (e.g., CD137(4-1BB)); host cell proliferation; or APC killing (e.g., using a labeled chromium release assay).

[0061] As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotide, polynucleotide, any of their fragments generated, for example, by polymerase chain reaction (PCR) or in vitro translation, and also fragments generated by either ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modifications in, or replacements 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 phosphorothioate, phosphorodithioate, phosphonoselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, etc. Nucleic acid monomers can contain phosphorothioate, phosphorodithioate, or phosphoroselenoate linkages, or any combination thereof. Nucleic acid molecules can be either single-stranded or double-stranded.

[0062] 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 coexisting materials in the natural system is isolated. Such a nucleic acid may be part of a vector, and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), and such a vector or composition may still be isolated in that it is not part of the natural environment for the nucleic acid or polypeptide. In some embodiments, isolated binding proteins, polynucleotides, vectors, or host cells are provided. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain, including regions preceding and following the coding region ("leader and trailer"), as well as intervening sequences (introns) between individual coding segments (exons).

[0063] 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 the introduction of an exogenous nucleic acid molecule, or to a cell or microorganism that has been genetically engineered by human intervention, i.e., by the introduction of a heterologous nucleic acid molecule, or to a cell or microorganism in which the expression of an endogenous nucleic acid molecule or gene has been altered so that it is regulated, deregulated, or constitutive; such an alteration or modification may be introduced by genetic engineering. Artificially produced genetic alterations can include, for example, modifications that introduce nucleic acid molecules (which may include expression control elements such as promoters) encoding one or more proteins or enzymes, or the addition, deletion, or substitution of other nucleic acid molecules, or other functional disruption or addition to a cell's genetic material. Exemplary modifications include modifications in the coding region of a heterologous or homologous polypeptide or functional fragment thereof from a reference or parent molecule.

[0064] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of changes in the sequence, including substitution, insertion, or deletion of nucleotide(s) or amino acid(s). In certain embodiments, the mutation is a substitution of one or three codons or amino acids, a deletion of one to about five codons or amino acids, or a combination thereof.

[0065] A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO97 / 09433, p. 10; Lehninger, Biochemistry, 2002). nd Edition, Worth Publishers, Inc. NY, NY, pp. 71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0066] In certain embodiments, proteins (e.g., binding proteins, immunogenic peptides) in accordance with the present disclosure comprise variant sequences compared to a reference sequence (e.g., a variant TCR CDR (e.g., CDR3β) compared to a reference TCR CDR disclosed herein). CDRs (e.g., CDR3β). As used herein, a "variant" amino acid sequence, peptide, or polypeptide can refer to an amino acid sequence (or peptide or polypeptide) that has one, two, or three amino acid substitutions, deletions, or insertions compared to a reference amino acid sequence. In certain embodiments, a variant amino acid sequence, peptide, or polypeptide maintains substantially the same functionality (e.g., binding specificity and affinity to a peptide:HLA complex) as the reference molecule; for example, a variant TCR fragment disclosed herein maintains 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 compared to a reference TCR-binding fragment.

[0067] An "altered domain" or "altered protein" refers to a motif, region, domain, peptide, polypeptide, or protein that has 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%, at least 99.9%) non-identical sequence identity with a wild-type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain, TCR beta constant domain).

[0068] Altered domains or proteins or derivatives can be based on all possible codon choices for the same amino acid and conservative amino acid substitutions. For example, the following six groups each contain amino acids that are conservative substitutions for each other: 1) alanine (ala; A), serine (ser; S), threonine (thr; T); 2) aspartic acid (asp; D) and glutamic acid (glu; E); 3) asparagine (asn; N) and glutamine (gln; 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). (WO 97 / 09433, p. 10, Lehninger, Biochemistry, 2009, pp. 111-114, 2011). nd (See also "Conservative Substitutions," 1984, 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."

[0069] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule. A "transgene" or "transgene construct" refers to a construct containing two or more genes operably linked in an arrangement 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 such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with each other so that the function of one does not affect the other. In some embodiments, a gene present in a transgene is operably linked to an expression control sequence (e.g., a promoter).

[0070] The construct (e.g., transgene) can be present in a vector (e.g., bacterial vector, viral vector) or can be integrated into a genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. Vectors can include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules, for example, plasmids, cosmids, viruses, RNA vectors, or linear or circular DNA or RNA molecules. Exemplary vectors are those capable of autonomous replication (episomal vectors) or those capable of expressing nucleic acid molecules linked to them (expression vectors). Vectors useful in the compositions and methods of the present disclosure are further described herein.

[0071] The term "expression," as used herein, refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule, such as a gene. This process can include transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof.

[0072] 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, where the nucleic acid molecule can be integrated into the genome of the cell (e.g., a chromosome, a plasmid, a plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., a transfected mRNA).

[0073] As used herein, a "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 the host cell but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule derived from the host cell. The source of a heterologous or exogenous nucleic acid molecule, construct, or sequence may be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule is added to a host cell or host genome (i.e., is not endogenous or native) by, for example, conjugation, transformation, transfection, transduction, electroporation, etc., and the added molecule can be integrated into the host genome or can exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can exist in multiple copies. Additionally, "heterologous" refers to a non-native enzyme, protein, or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity. Furthermore, a cell containing a "modified" or "heterologous" polynucleotide or binding protein includes the progeny of that cell, whether or not the progeny itself has been transduced, transfected, or otherwise manipulated or altered.

[0074] 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 multiple individually regulated 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 molecules encoding a desired TCR (e.g., TCRα and TCRβ) specific for a Ras antigenic peptide, and optionally also encoding a CD8 co-receptor polypeptide, including an α chain, a β chain, or portions thereof, e.g., an extracellular portion capable of binding to MHC, as disclosed herein. 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., in a single vector), on separate vectors, and can be integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities referenced refers to the number of encoding nucleic acid molecules or protein activities, and not necessarily the number of separate nucleic acid molecules introduced into the host cell.

[0075] As used herein, the terms "endogenous" or "native" refer to a gene, protein, or activity that is normally present in a host cell. Furthermore, a gene, protein, or activity that is mutated, overexpressed, shuffled, duplicated, or otherwise altered compared to a parent gene, protein, or activity is still considered endogenous or native to that particular host cell. For example, an endogenous control sequence (e.g., promoter, translation attenuation sequence) from a first gene can be used to alter or regulate the expression of a second, native gene or nucleic acid molecule, where the expression or regulation of the second, native gene or nucleic acid molecule differs from its normal expression or regulation in the parent cell.

[0076] The terms "homologous" or "homolog" refer 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, optionally, can have altered expression levels, a different sequence, an altered activity, or any combination thereof.

[0077] "Sequence identity," as used herein, refers to the percentage of amino acid residues or nucleic acid bases (respectively) in a sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Percentage sequence identity values can be generated using the NCBI BLAST 2.0 software defined by Altschul et al. (1997), Nucl. Acids Res. 25:3389-3402, with parameters set to default. Additionally or alternatively, the degree of sequence identity between two sequences can be determined by comparing the two sequences using a computer program designed for this purpose, such as a global or local alignment algorithm. 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 the Needleman and Wunsch algorithm can be used to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Default settings can be used.

[0078] To generate a similarity score for two amino acid sequences, a scoring matrix can be used that assigns positive scores to some non-identical amino acids (e.g., conservative amino acid substitutions, amino acids with similar physiochemical properties, and / or amino acids that show frequent substitutions in orthologs, homologs, or paralogs). Non-limiting examples of scoring matrices include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0079] Variants of the nucleic acid molecules of the present disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identical to a nucleic acid molecule of a defined or reference polynucleotide described herein, or hybridize to the polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the ability to encode a binding protein or binding domain thereof having the functionality described herein, such as binding to a target molecule.

[0080] 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 materials with which it is present in the natural system is isolated. Such a nucleic acid may be part of a vector, and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), and such a vector or composition may still be isolated in that it is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" means a segment of DNA involved in producing a polypeptide chain, including regions preceding and following the coding region ("leader and trailer"), as well as intervening sequences (introns) between individual coding segments (exons).

[0081] In some contexts, the term "variant," as used herein, refers to at least a fragment of a reference full-length sequence, more specifically, one or more amino acid or nucleic acid sequences that are truncated at one or both ends by one or more amino acids compared to the full-length sequence. Such fragments comprise or encode peptides having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, or 200 consecutive amino acids of the original sequence or its variant. The full length of a variant can be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids.

[0082] In some embodiments, the term "variant" refers to at least one fragment, as well as to a polypeptide or fragment thereof comprising an amino acid sequence that is 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 or fragment thereof referred to, in which amino acids other than those essential for biological activity or for folding 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 so as to preserve the biological activity of the polypeptide. The state of the art includes various methods that can be used to align two given nucleic acid or amino acid sequences and calculate the degree of identity (see, for example, Arthur Lesk (2008), Introduction to bioinformatics, Oxford University Press, 2008, 3rd edition). In some embodiments, Clustal W software can be used using default settings (Larkin, MA, et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948).

[0083] In certain embodiments, variants may additionally comprise chemical modifications, e.g., isotope labeling or covalent modifications such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, etc. Methods for modifying polypeptides are known and are generally used so as not to eliminate or substantially reduce the desired activity of the polypeptide.

[0084] In one embodiment, the term "variant" of a nucleic acid molecule includes, for example, a nucleic acid of a complementary strand that hybridizes to a reference nucleic acid or wild-type nucleic acid under stringent conditions. The stringency of a hybridization reaction can be readily determined by one of ordinary skill in the art and is generally an empirical calculation dependent on the length of the probe, washing temperature, and salt concentration. Longer probes generally require higher temperatures for proper annealing, while shorter probes require lower temperatures. 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 desired degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Consequently, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures tend to be less so. For further details and explanation of the stringency of hybridization reactions, see Ausubel, FM (1995), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. Furthermore, those skilled in the art can follow the instructions in the manuals 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, KH (1991) International Journal of Systematic Bacteriology 41:255-260, regarding methods for identifying DNA sequences by hybridization. In one embodiment, stringent conditions are applied to any hybridization. That is, hybridization occurs only if the probe is 70% or more identical to the target sequence.Probes with a lower degree of identity to the target sequence may hybridize, but such hybrids will be unstable and will be removed under stringent conditions, e.g., by reducing the salt concentration to 2×SSC, or optionally followed by a wash step to 0.5×SSC, while the temperature is, for example, about 50°C to 68°C, about 52°C to 68°C, about 54°C to 68°C, about 56°C to 68°C, about 58°C to 68°C, about 60°C to 68°C, about 62°C to 68°C, about 64°C to 68°C, or about 66°C to 68°C. The salt concentration can be adjusted to 0.2×SSC or even 0.1×SSC. Nucleic acid sequences having a degree of identity to a 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% can be isolated. In one 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 as a reference nucleic acid sequence and variants thereof, in accordance with the degeneracy of the genetic code.

[0085] "Functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of the present disclosure, but that in some contexts differs 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 able to perform 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% of the level of the activity of the parent polypeptide. In other words, a functional variant of a polypeptide of the disclosure or of the encoded polypeptide has "similar binding," "similar affinity," or "similar activity" if the functional variant exhibits no more than a 50% decrease in performance in a selected assay compared to the parent or reference polypeptide, such as an assay to measure binding affinity (e.g., Biacore® or tetramer staining to measure association (Ka) or dissociation (KD) constants), avidity, or host cell activation. 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, wherein the polypeptide or encoded polypeptide retains at least 50% of the 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% of the level of the parent polypeptide or provides a biological benefit (e.g., effector function).

[0086] A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure has "similar binding" or "similar activity" if the functional portion or fragment exhibits no more than a 50% decrease in performance (preferably no more than a 20% or 10% or log difference compared to the parent or reference for affinity) in a selected assay compared to a parent or reference polypeptide, such as an assay for measuring binding affinity or for measuring effector function (e.g., cytokine release). Functional variants of the specifically disclosed binding proteins and polynucleotides are contemplated.

[0087] An "altered domain" or "altered protein" refers to a motif, region, domain, peptide, polypeptide, or protein that has 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%, at least 99.9%) non-identical sequence identity with a wild-type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain, or TCR beta constant domain).

[0088] Binding proteins In one aspect, the disclosure provides a binding protein comprising a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain, wherein the binding protein is capable of binding to a peptide:HLA complex, and wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3. In certain embodiments, the HLA comprises HLA-A*11, optionally HLA-A*11:01. In any of the embodiments disclosed herein, the binding protein may be heterologously expressed by human immune system cells, such as T cells.

[0089] In certain embodiments, the Vα domain and / or Vβ domain are each independently human, humanized, or chimeric, preferably each human. In some embodiments, the Vα domain is human and the Vβ domain is human. The binding proteins, compositions, and methods disclosed herein can utilize Vα domains, Vβ domains, or CDRs derived therefrom derived from a human subject, for example, CDRs derived from sequencing T cells isolated from a human subject or a population thereof. TCR Vα domains, Vβ 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, Vα domains, Vβ domains, and CDRs derived from a human subject can undergo negative thymic selection against substantially the entire human peptidome presented by the complete set of human HLA molecules in vivo, which can reduce the likelihood of the binding protein being cross-reactive with other human self-antigens.

[0090] In some embodiments, the binding proteins disclosed herein are substantially non-reactive with human proteomes presented by one or more HLA alleles disclosed herein, e.g., one or any combination of HLA alleles from Table 3. Reactivity can be determined by any suitable method, such as those disclosed in Examples 5-7 and 13 of the present application. In some embodiments, no significant response due to binding of protein-transduced T cells to human proteomes presented by one or more HLA allele(s) is observed or predicted at peptide concentrations of 500 nM or less, 400 nM or less, 300 nM or less, 200 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. In some embodiments, the binding proteins disclosed herein are substantially non-reactive with peptide:HLA complexes, wherein the peptide comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 118-148. In some embodiments, the HLA comprises HLA-A*11. In a further embodiment, the HLA comprises HLA-A*11:01.

[0091] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from (e.g., identified) a T cell of a subject (e.g., a human subject) having a disease such as cancer. In some embodiments, the 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 as disclosed herein. In some embodiments, the 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 G12 mutation, such as a KRAS G12V or G12D mutation. In some embodiments, the 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 cells comprising a KRAS G12 mutation, such as a KRAS G12V or G12D mutation.

[0092] In some embodiments, the 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, the healthy subject lacks a particular pathological diagnosis (e.g., a disease diagnosis such as a cancer diagnosis). In some embodiments, the healthy subject lacks a particular pathological diagnosis but includes a different pathological diagnosis, for example, lacking a cancer diagnosis but including a diagnosis of hypertension or type II diabetes.

[0093] The binding proteins disclosed herein can be heterologously expressed by a host cell, e.g., a human immune cell, such as a T cell. Furthermore, expression of the binding proteins disclosed herein can confer advantageous properties to the host cell, such as binding specificity for the disclosed Ras antigen:HLA complex, improved activation, proliferation, or killing activity in the presence of Ras antigen:HLA-presenting tumor cells.

[0094] 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, an NK cell, or an NK-T cell), the immune cell is an HLA-A*11:01 T cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 3. + It is possible to specifically kill tumor cells. Killing of target cells can be determined, for example, with the Incucyte® bioimaging platform (Essen Bioscience). In certain embodiments, this platform uses activated caspases and labeled (e.g., RapidRed or NucRed) tumor cell signals, where overlap is measured and increased overlap area is equivalent to tumor cell death by apoptosis. Killing is determined by the incorporation of labeled chromium ( 51 Cr) was loaded, and the supernatant 51Cr can also be determined using a 4-hour assay, in which Cr is measured, for example, after 4 hours of co-incubation with immune cells expressing a binding protein of the disclosure. In certain embodiments, killing assays can be performed using effector:target cell ratios such as 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.

[0095] In any of the embodiments disclosed herein, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, an NK cell, or an NK-T cell), the immune cell is an HLA-A11:01 T cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 3. + In the presence of tumor cells, optionally in the further presence of exogenous IFN-γ, expression of Nur77 is elevated, wherein Nur77 expression is elevated compared to (i) Nur77 expression by a reference immune cell that does not express the binding protein (i.e., of the same cell type as the immune cell that expresses the binding protein, and if not, a cell type that is at least substantially identical or functionally equivalent phenotypically and / or genotypically) when the reference immune cell is in the presence of tumor cells; and / or (ii) Nur77 expression by an immune cell that expresses the binding protein (wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO:2 or 3, and the HLA is optionally HLA-A*11:01) when not in the presence of tumor cells and / or in the absence of antigen-presenting cells that express the peptide:HLA complex. Expression of Nur77 can be determined, for example, using a reporter construct; for example, a transgenic expression construct comprising the Nur77 locus operably linked to a sequence encoding dTomato (see Ahsouri and Weiss, J Immunol 198(2):657-668 (2017)).

[0096] In any of the embodiments disclosed herein, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, an NK cell, or an NK-T cell), the immune cell is an HLA-A*02 T cell that expresses a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 3. + In the presence of tumor cells, optionally in the further presence of exogenous IFN-γ, expression of CD137 (also known as 4-1BB) is elevated, and CD137 expression is elevated compared to (i) CD137 expression by reference immune cells that do not express the binding protein when the reference immune cells are in the presence of tumor cells; and / or (ii) CD137 expression by immune cells that express the binding protein (wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 2 or 3, and the HLA is optionally HLA-A*11:01) in the absence of tumor cells and / or in the absence of antigen-presenting cells that express the peptide:HLA complex. CD137 expression can be determined, for example, using flow cytometry using a labeled anti-CD137 antibody. In certain embodiments, CD137 is measured after a 16-hour assay in which immune cells are co-incubated with or stimulated by the peptide or target cells expressing the peptide.

[0097] In any of the embodiments disclosed herein, (i) the binding protein is encoded by a polynucleotide heterologous to the immune cell, and (ii) the immune cell is a human CD8 + and (iii) tumor cells expressing a peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 2 or 3 are HLA-A*11:01. + and / or (iv) the tumor cells comprise OVCAR5 (ovarian serous adenocarcinoma), DAN-G (pancreatic adenocarcinoma), CFPAC1 (pancreatic adenocarcinoma), SW480 (colon cancer), SW527 (breast cancer), or NCI-H441 (lung adenocarcinoma) cells.

[0098] In certain embodiments, the binding protein is capable of binding to peptide:HLA complexes independently of, or in the absence of, CD8. CD8-independent binding is observed in CD8-negative cells (e.g., CD4 +

[0013] The binding of the target can be determined by expressing the binding protein in T cells (e.g., T cells, Jurkat cells, etc.) and identifying binding of the cells to the target. In some embodiments, a binding protein is provided comprising: (a) a T cell receptor (TCR) alpha chain variable (Vα) domain comprising a complementarity determining region 3 (CDR3α) amino acid sequence set forth in any one of SEQ ID NOs: 16, 17, 42, and 43, or a variant thereof, optionally with one, two, or three conservative amino acid substitutions, and / or (b) a TCR beta chain variable (Vβ) domain comprising a CDR3β amino acid sequence set forth in any one of SEQ ID NOs: 26, 27, 52, and 53, or a variant thereof, optionally with one, two, or three conservative amino acid substitutions, wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3), and the HLA comprises HLA-A*11. In certain embodiments, the HLA comprises HLA-A*11:01. The binding protein can comprise a Vα domain and a Vβ domain.

[0099] The Vα and / or Vβ domains may be human, humanized, or chimeric, preferably human.

[0100] In certain embodiments, the binding protein comprises the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: (i) 17 and 27, respectively, or variants thereof, optionally with one, two, or three conservative amino acid substitutions; (ii) 16 and 26, respectively, or variants thereof, optionally with one, two, or three conservative amino acid substitutions; (iii) 53 and 43, respectively, or variants thereof, optionally with one, two, or three conservative amino acid substitutions; or (iv) 52 and 42, respectively, or variants thereof, optionally with one, two, or three conservative amino acid substitutions. In certain embodiments, the binding protein comprises the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: (i) 17 and 27, respectively; (ii) 16 and 26, respectively; (iii) 53 and 43, respectively; or (iv) 52 and 42, respectively.

[0101] In some embodiments, the binding protein comprises (i) in the Vα domain, the CDR1α amino acid sequence set forth in SEQ ID NO: 14 or 40, or a variant thereof, optionally with one or two conservative amino acid substitutions; (ii) in the Vα domain, the CDR2α amino acid sequence set forth in SEQ ID NO: 15 or 41, or a variant thereof, optionally with one or two conservative amino acid substitutions; (iii) in the Vβ domain, the CDR1β amino acid sequence set forth in SEQ ID NO: 24 or 50, or a variant thereof, optionally with one or two conservative amino acid substitutions; (iv) in the Vβ domain, the CDR2β amino acid sequence set forth in SEQ ID NO: 25 or 51, or a variant thereof, optionally with one or two conservative amino acid substitutions; or (v) any combination of (i)-(iv).

[0102] In some embodiments, the binding protein further comprises (i) in the Vα domain, a CDR1α amino acid sequence set forth in SEQ ID NO: 14 or 40; (ii) in the Vα domain, a CDR2α amino acid sequence set forth in SEQ ID NO: 15 or 41; (iii) in the Vβ domain, a CDR1β amino acid sequence set forth in SEQ ID NO: 24 or 50; (iv) in the Vβ domain, a CDR2β amino acid sequence set forth in SEQ ID NO: 25 or 51; or (v) any combination of (i)-(iv).

[0103] In some embodiments, the binding protein further comprises: (i) in the Vα domain, a CDR1α amino acid sequence set forth in SEQ ID NO: 14 or 40; (ii) in the Vα domain, a CDR2α amino acid sequence set forth in SEQ ID NO: 15 or 41; (iii) in the Vβ domain, a CDR1β amino acid sequence set forth in SEQ ID NO: 24 or 50; and (iv) in the Vβ domain, a CDR2β amino acid sequence set forth in SEQ ID NO: 25 or 51.

[0104] In certain embodiments, the binding protein comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 14, 15, 16 or 17, 24, 25, and 26 or 27, respectively.

[0105] In other embodiments, the binding protein comprises the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 40, 41, 42 or 43, 50, 51, and 52 or 52, respectively.

[0106] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by the Kabat method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 49, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Kabat method from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by the Kabat method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Kabat method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β identified by the Kabat method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the Kabat method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β identified by the Kabat method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Kabat method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β identified by the Kabat method from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the Kabat method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β identified by the Kabat method from the variable domain sequence of SEQ ID NO: 49.

[0107] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by the Chothia method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 49, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Chothia method from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by the Chothia method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β identified by the Chothia method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β identified by the Chothia method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β identified by the Chothia method from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β identified by the Chothia method from the variable domain sequence of SEQ ID NO: 49.

[0108] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by EU law or numbering scheme from the variable domain sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 49, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by EU law from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by EU law from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by EU methods from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β identified by EU methods from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by EU methods from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β identified by EU methods from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by EU methods from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β identified by EU methods from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by EU methods from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β identified by EU methods from the variable domain sequence of SEQ ID NO: 49.

[0109] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by the IMGT method or numbering scheme (including IMGT and / or IMGT junctions for CDR3) from the variable domain sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 49, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the IMGT method from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by the IMGT method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the IMGT method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β identified by the IMGT method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the IMGT method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β identified by the IMGT method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the IMGT method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β identified by the IMGT method from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the IMGT method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β identified by the IMGT method from the variable domain sequence of SEQ ID NO: 49.

[0110] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by the IMGT method or numbering scheme (including IMGT and / or IMGT junctions for CDR3) from the amino acid sequence set forth in SEQ ID NO: 20, the amino acid sequence set forth in SEQ ID NO: 30, the amino acid sequence set forth in SEQ ID NO: 155, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the IMGT method from the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by the IMGT method from the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 155. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the IMGT method from the amino acid sequence set forth in SEQ ID NO: 20, and CDR1β, CDR2β, and CDR3β identified by the IMGT method from the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 155.

[0111] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by the extended Chothia method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 49, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β identified by the extended Chothia method from the variable domain sequence of SEQ ID NO: 49.

[0112] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β identified by the Aho method or numbering scheme from the variable domain sequence of SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 39, SEQ ID NO: 49, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Aho method from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β identified by the Aho method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Aho method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β identified by the Aho method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the Aho method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β identified by the Aho method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α identified by the Aho method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β identified by the Aho method from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α identified by the Aho method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β identified by the Aho method from the variable domain sequence of SEQ ID NO: 49.

[0113] In some embodiments, the binding protein comprises a CDR1α that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14 or 40, or a CDR1α sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39.

[0114] In some embodiments, the binding protein comprises a CDR2α that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 15 or 41, or a CDR2α sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39.

[0115] In some embodiments, the binding protein comprises a CDR3α that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 16, 17, 42, or 43, or a CDR3α sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39.

[0116] In some embodiments, the binding protein comprises a CDR1β that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 24 or 50, or a CDR1β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49.

[0117] In some embodiments, the binding protein comprises a CDR2β that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 25 or 51, or a CDR2β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49.

[0118] In some embodiments, the binding protein comprises a CDR3β that comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 26, 27, 52, or 53, or a CDR3β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49.

[0119] In some embodiments, the binding protein comprises a CDR1α that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 14 or 40, or a CDR1α sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 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 consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0120] In some embodiments, the binding protein comprises a CDR2α that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 15 or 41, or a CDR2α sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 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 consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0121] In some embodiments, the binding protein comprises a CDR3α that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 16, 17, 42, or 43, or a CDR3α sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 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 consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0122] In some embodiments, the binding protein comprises a CDR1β that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 24 or 50, or a CDR1β sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. 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 consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0123] In some embodiments, the binding protein comprises a CDR2β that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 25 or 51, or a CDR2β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. 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 consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0124] In some embodiments, the binding protein comprises a CDR3β that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 26, 27, 52, or 53, or a CDR3β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. 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 consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0125] In some embodiments, the binding protein comprises a CDR1α that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 14 or 40, or a CDR1α sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 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.

[0126] In some embodiments, the binding protein comprises a CDR2α that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 15 or 41, or a CDR2α sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 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.

[0127] In some embodiments, the binding protein comprises a CDR3α that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 16, 17, 42, or 43, or a CDR3α sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 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.

[0128] In some embodiments, the binding protein comprises a CDR1β that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 24 or 50, or a CDR1β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. 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.

[0129] In some embodiments, the binding protein comprises a CDR2β that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 25 or 51, or a CDR2β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. 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.

[0130] In some embodiments, the binding protein comprises a CDR3β that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 26, 27, 52, or 53, or a CDR3β sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. 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.

[0131] The binding proteins disclosed herein may comprise one or more framework regions (FRs). For example, a binding protein may comprise a variable domain comprising three CDRs and four FRs, or two variable domains each comprising three CDRs and four FRs. Exemplary FR amino acid sequences are provided by SEQ ID NOs: 91-117 and 153. The framework regions used in the binding proteins may be mammalian framework regions. The framework regions used in the binding proteins may be human framework regions. The framework regions used in the binding proteins may be engineered framework regions.

[0132] The binding protein may comprise FR1, FR2, and FR3, and / or FR4 disclosed herein. In some embodiments, the binding protein comprises a Vα comprising an FR1 that comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 91, 103, and 115, or a variant thereof; an FR2 that comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 92, 104, or a variant thereof; an FR3 that comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 93, 95, 105, and 107, or a variant thereof; and an FR4 that comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 94, 96, 106, and 108, or a variant thereof. In some embodiments, the binding protein comprises a Vα that comprises the amino acid sequence set forth in SEQ ID NO: 115. In some embodiments, the binding protein comprises a Vα that comprises the amino acid sequence set forth in SEQ ID NO: 91.

[0133] In some embodiments, the binding protein comprises a Vβ comprising an FR1 that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 97, 109, and 153, or a variant thereof; an FR2 that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 98, 110, or a variant thereof; an FR3 that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 99, 101, 111, 113, or a variant thereof; and an FR4 that comprises, consists essentially of, or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 100, 102, 112, 114, 116, and 117, or a variant thereof.

[0134] In some embodiments, the binding protein comprises a Vβ comprising the amino acid sequence set forth in SEQ ID NO: 153. In some embodiments, the binding protein comprises a Vβ comprising the amino acid sequence set forth in SEQ ID NO: 97.

[0135] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as identified by the Kabat method or numbering scheme from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding protein comprises a Vβ domain comprising FR1, FR2, FR3, and FR4 as identified by the Kabat method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0136] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as identified by the Chothia method or numbering scheme from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding protein comprises a Vβ domain comprising FR1, FR2, FR3, and FR4 as identified by the Chothia method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0137] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as identified by EU law or numbering scheme from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding protein comprises a Vβ domain comprising FR1, FR2, FR3, and FR4 as identified by EU law from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0138] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 identified by the IMGT method or numbering scheme from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding protein comprises a Vβ domain comprising FR1, FR2, FR3, and FR4 identified by the IMGT method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0139] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 identified by the extended Chothia numbering scheme from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding protein comprises a Vβ domain comprising FR1, FR2, FR3, and FR4 identified by the extended Chothia numbering scheme from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0140] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as identified by the Aho method or numbering scheme from the variable domain sequence of SEQ ID NO: 13 or SEQ ID NO: 39. In some embodiments, the binding protein comprises a Vβ domain comprising FR1, FR2, FR3, and FR4 as identified by the Aho method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0141] In some embodiments, the binding protein comprises a Vα domain comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 91, 103, or 115, or an FR1 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39.

[0142] In some embodiments, the binding protein comprises a Vα domain comprising an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 92 or 104, or an FR2 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39.

[0143] In some embodiments, the binding protein comprises a Vα domain comprising an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 93, 95, 105, or 107, or an FR3 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39.

[0144] In some embodiments, the binding protein comprises a Vα domain comprising an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 94, 96, 106, or 108, or an FR4 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39.

[0145] In some embodiments, the binding protein comprises a Vβ domain comprising an FR1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 97 or 109, or an FR1 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49.

[0146] In some embodiments, the binding protein comprises a Vβ domain comprising an FR2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 98 or 110, or an FR2 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49.

[0147] In some embodiments, the binding protein comprises a Vβ domain comprising an FR3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 99, 101, 111, or 113, or an FR3 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49.

[0148] In some embodiments, the binding protein comprises a Vβ domain comprising an FR4 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 100, 102, 112, 114, 116, or 117, or an FR4 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49.

[0149] In some embodiments, the binding protein comprises a Vα domain comprising an FR1 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 91, 103, or 115, or an FR1 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0150] In some embodiments, the binding protein comprises a Vα domain comprising an FR2 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 92 or 104, or an FR2 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0151] In some embodiments, the binding protein comprises a Vα domain comprising an FR3 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 93, 95, 105, or 107, or an FR3 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0152] In some embodiments, the binding protein comprises a Vα domain comprising an FR4 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 94, 96, 106, or 108, or an FR4 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0153] In some embodiments, the binding protein comprises a Vβ domain comprising an FR1 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 97, or 109, or 153, or an FR1 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0154] In some embodiments, the binding protein comprises a Vβ domain comprising an FR2 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 98 or 110, or an FR2 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0155] In some embodiments, the binding protein comprises a Vβ domain comprising an FR3 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 99, 101, 111, or 113, or an FR3 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0156] In some embodiments, the binding protein comprises a Vβ domain comprising an FR4 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 100, 102, 112, 114, 116, or 117, or an FR4 sequence identified by the Kabat, Chothia, EU, IMGT, Extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The substitution(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be consecutive, non-consecutive, or a combination thereof. In some embodiments, the substitutions are conservative.

[0157] In some embodiments, the binding protein comprises a Vα domain comprising an FR1 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 91, 103, or 115, or an FR1 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0158] In some embodiments, the binding protein comprises a Vα domain comprising an FR2 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 92 or 104, or an FR2 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0159] In some embodiments, the binding protein comprises a Vα domain comprising an FR3 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 93, 95, 105, or 107, or an FR3 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 13 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0160] In some embodiments, the binding protein comprises a Vα domain comprising an FR4 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 94, 96, 106, or 108, or an FR4 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0161] In some embodiments, the binding protein comprises a Vβ domain comprising an FR1 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 97, 109, or 153, or an FR1 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0162] In some embodiments, the binding protein comprises a Vβ domain comprising an FR2 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 98 or 110, or an FR2 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0163] In some embodiments, the binding protein comprises a Vβ domain comprising an FR3 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 99, 101, 111, or 113, or an FR3 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0164] In some embodiments, the binding protein comprises a Vβ domain comprising an FR4 that comprises at most 1, at most 2, at most 3, at most 4, at most 5, or at most 6 amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 100, 102, 112, 114, 116, or 117, or an FR4 sequence identified by the Kabat, Chothia, EU, IMGT, extended Chothia, or Aho method from the variable domain of SEQ ID NO: 23 or 49. The insertion(s) and / or deletion(s) can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof.

[0165] The binding protein can comprise a TCRα FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, a TCRβ FR1, CDR1, FR2, CDR2, FR3, CDR3, or FR4 region, or a combination thereof.

[0166] In some embodiments, (i) the Vα 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 or 39; and / or (ii) the Vβ 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: 23 or 154 or 49.

[0167] In some embodiments, the Vα 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. and wherein the Vβ 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: 23 or 154.

[0168] In some embodiments, the Vα 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 similarity to the amino acid sequence set forth in SEQ ID NO:39, and the Vβ 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 similarity to the amino acid sequence set forth in SEQ ID NO:49.

[0169] In certain embodiments, the Vα domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the binding protein comprises a TCR β chain, wherein the TCR β chain comprises the amino acid KA immediately N-terminal to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the Vα domain comprises the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain comprises the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the Vα domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the Vα domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 23.

[0170] In some embodiments, a binding protein is provided comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 13 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 23 or 154.

[0171] In some embodiments, a binding protein capable of binding to a peptide:HLA complex is provided, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NO:2 or SEQ ID NO:3, and the HLA is optionally HLA-A*11, and further optionally HLA-A*11:01. 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 SEQ ID NO:13, and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:23 or 154. The first polypeptide may be or may comprise a TCR alpha chain, and / or the second polypeptide may be or may comprise a TCR beta chain. In some embodiments, the first polypeptide is or may comprise a TCR alpha chain, and / or the second polypeptide is or may comprise a TCR beta chain.

[0172] In certain embodiments, the Vα domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 39, and the Vβ domain comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the Vα domain comprises the amino acid sequence set forth in SEQ ID NO: 39, and the Vβ domain comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the Vα domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 39, and the Vβ domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the Vα domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 39, and the Vβ domain consists essentially of the amino acid sequence set forth in SEQ ID NO: 49.

[0173] In some embodiments, a binding protein is provided comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 13 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 23 or 154.

[0174] In some embodiments, a binding protein is provided comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 20 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 30.

[0175] In some embodiments, a binding protein is provided comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain comprises the amino acid sequence set forth in SEQ ID NO: 20 and the TCR beta chain comprises the amino acid sequence set forth in SEQ ID NO: 155.

[0176] In some embodiments, a binding protein capable of binding to a peptide:HLA complex is provided, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NO:2 or SEQ ID NO:3, and the HLA is optionally HLA-A*11, and further optionally HLA-A*11:01. 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 SEQ ID NO:20 and the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO:155. The first polypeptide may be or may comprise a TCR alpha chain, and / or the second polypeptide may be or may comprise a TCR beta chain. In some embodiments, the first polypeptide is or may comprise a TCR alpha chain, and / or the second polypeptide is or may comprise a TCR beta chain.

[0177] In some embodiments, the variable domain comprises an amino acid sequence having one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0178] For example, the variable domain can comprise an amino acid sequence having an insertion of 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 acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0179] In some embodiments, the variable domain comprises an amino acid sequence with an insertion of 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 acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0180] In some embodiments, the variable domain comprises an insertion of 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 acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0181] The one or more insertions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions may be contiguous, non-contiguous, or a combination thereof.

[0182] In some embodiments, the variable domain comprises an amino acid sequence having 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, 23, 39, and 49.

[0183] In some embodiments, the variable domain comprises an amino acid sequence having a deletion of 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 acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0184] In some embodiments, the variable domain comprises a deletion of 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 acids relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0185] The one or more deletions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, non-contiguous, or a combination thereof.

[0186] In some embodiments, the variable domain comprises an amino acid sequence having 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: 13, 23, 39, and 49.

[0187] 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: 13, 23, 39, and 49.

[0188] 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: 13, 23, 39, and 49.

[0189] The one or more substitutions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions may be consecutive, non-consecutive, or a combination thereof.

[0190] The binding protein may further comprise a TCR alpha chain constant domain (Cα) and / or a TCR beta chain constant domain (Cβ). The TCR alpha chain constant domain (Cα) and / or the TCR beta chain constant domain (Cβ) may be human. The TCR alpha chain constant domain (Cα) and / or the TCR beta chain constant domain (Cβ) may be mammalian. The TCR alpha chain constant domain (Cα) and / or the TCR beta chain constant domain (Cβ) may be an engineered variant of a mammalian (e.g., human) constant domain. In some embodiments, the Cα is an engineered variant of human Cα and / or the Cβ is an engineered variant of human Cβ. In some embodiments, the Cα is an engineered variant of human Cα and the Cβ is an engineered variant of human Cβ.

[0191] In some embodiments, Cα comprises, consists essentially of, or consists of an amino acid sequence having 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%, or at least 99% identity to, comprising, or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 44, 45, and 69.

[0192] In some embodiments, Cβ comprises, consists essentially of, or consists of an amino acid sequence having at least 70%, 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%, 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.

[0193] In some embodiments, Cα and Cβ comprise or consist of an amino acid sequence having 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%, or at least 99% identity to, comprising, or consisting of the amino acid sequence 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.

[0194] The binding protein can comprise (i) the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain, (ii) the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain, and / or (iii) the cytoplasmic domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. The binding protein can comprise a full-length or substantially full-length TCR alpha chain, a TCR beta chain, a TCR gamma chain, and / or a TCR delta chain.

[0195] In some embodiments, the binding protein comprises a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain are selected from the group consisting of: (i) the amino acid sequence set forth in SEQ ID NOs: 12 and 22, respectively; (ii) the amino acid sequence set forth in SEQ ID NOs: 20 and 30, respectively; (iii) the amino acid sequence set forth in SEQ ID NOs: 12 and 30, respectively; (iv) the amino acid sequence set forth in SEQ ID NOs: 20 and 22, respectively; (v) the amino acid sequence set forth in SEQ ID NOs: 38 and 48, respectively; (vi) the amino acid sequence set forth in SEQ ID NOs: 46 and 56, respectively; (vii) the amino acid sequence set forth in SEQ ID NOs: 12 and 30, respectively; or (viii) an amino acid sequence having 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%, or at least 99% identity to, comprising, or consisting of, the amino acid sequence set forth in SEQ ID NOs: 38 and 56, respectively; or (viii) the amino acid sequence set forth in SEQ ID NOs: 46 and 48, respectively.

[0196] In some embodiments, a first polypeptide and a second polypeptide are provided, wherein (i) the first polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 83, and (ii) the second polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 85, and the first polypeptide and the second polypeptide are capable of associating to form a polypeptide dimer.

[0197] In some embodiments, the binding protein comprises an amino acid sequence having one or more insertions, deletions, and / or substitutions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0198] For example, the binding protein can comprise an amino acid sequence having an insertion of 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 acids relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0199] In some embodiments, the binding protein comprises an amino acid sequence with an insertion of 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 acids relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0200] In some embodiments, the binding protein comprises an insertion of 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 acids relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0201] The one or more insertions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions may be contiguous, non-contiguous, or a combination thereof.

[0202] In some embodiments, the binding protein comprises an amino acid sequence having 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: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0203] In some embodiments, the binding protein comprises an amino acid sequence with a deletion of 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 acids relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0204] In some embodiments, the binding protein comprises a deletion of 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 acids relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0205] The one or more deletions can be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions can be contiguous, non-contiguous, or a combination thereof.

[0206] In some embodiments, the binding protein comprises an amino acid sequence having 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: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0207] In some embodiments, the binding protein comprises an amino acid sequence having 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: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0208] In some embodiments, the binding protein 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: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0209] The one or more substitutions may be at the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions may be consecutive, non-consecutive, or a combination thereof.

[0210] In any embodiment disclosed herein, the binding protein may comprise a TCR, a single-chain TCR (scTCR), a scTv, or a chimeric antigen receptor (CAR). Methods for producing engineered TCRs are described, for example, in Bowerman et al., Mol. Immunol., 46(15):3000 (2009), the techniques of which are incorporated herein by reference. Methods for producing 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 Application Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al., 2007, Clin. Cancer Res. 13:5426, the techniques of which are incorporated herein by reference. In some embodiments, the binding protein optionally comprises a soluble TCR fused to a binding domain (e.g., an scFv) specific for a CD3 protein. See Elie Dolgin, Nature Biotechnology 40:441-449 (2022).

[0211] In any of the embodiments disclosed herein, the polynucleotide encoding the binding protein may further comprise: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain, where optionally the encoded polypeptide is or comprises the CD8 co-receptor alpha chain; (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain, where optionally the encoded polypeptide is or comprises the CD8 co-receptor beta chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii). Without being bound by theory, in certain embodiments, co-expression or simultaneous expression of the binding protein and the CD8 co-receptor protein or portion thereof functional for binding to an HLA molecule results in increased activity of host cells (e.g., immune cells, e.g., T cells, optionally CD4 +The polynucleotide encoding the binding protein and the polynucleotide encoding the CD8 co-receptor polypeptide may be present on a single nucleic acid molecule (e.g., in the same expression vector) or may be present on separate nucleic acid molecules in the host cell.

[0212] In any embodiment of the present disclosure, the CD8 co-receptor alpha chain can comprise, consist essentially of, or consist of SEQ ID NO: 87, or SEQ ID NO: 87 from which the signal peptide has been removed. An example of a polynucleotide encoding SEQ ID NO: 87 is provided in SEQ ID NO: 88. In some embodiments, the CD8 co-receptor alpha chain comprises, consists essentially of, or consists of an amino acid sequence having 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: 87, or SEQ ID NO: 87 from which the signal peptide has been removed.

[0213] In any embodiment of the present disclosure, the CD8 co-receptor beta chain can comprise, consist essentially of, or consist of SEQ ID NO: 89, or SEQ ID NO: 89 from which the signal peptide has been removed. An example of a polynucleotide encoding SEQ ID NO: 89 is provided in SEQ ID NO: 90. In some embodiments, the CD8 co-receptor beta chain comprises, consists essentially of, or consists of an amino acid sequence having 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: 89, or SEQ ID NO: 89 from which the signal peptide has been removed.

[0214] In certain further embodiments, the polynucleotide comprises (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, (b) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain, and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b). In further embodiments, the polynucleotide comprises a polynucleotide encoding the self-cleaving peptide and disposed (1) between the polynucleotide encoding the binding protein and the polynucleotide encoding the polypeptide comprising the extracellular portion of the CD8 co-receptor α chain; and / or (2) between the polynucleotide encoding the binding protein and the polynucleotide encoding the polypeptide comprising the extracellular portion of the CD8 co-receptor β chain.

[0215] In still further embodiments, the polynucleotides are operably linked in-frame: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnBP), (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnBP), (iii) (pnBP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β), (iv) (pnBP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α), (v) (pnCD8α)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8β), or (vi) (pnCD8β)-(p The peptide may comprise (pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8α), where pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnBP is a polynucleotide encoding a binding protein, and pnSCP1 and pnSCP2 are each independently polynucleotides encoding a self-cleaving peptide, where these 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 the self-cleaving peptide can comprise a linker at its N-terminus and / or C-terminus. One example of a linker is GSG. In some embodiments, a T2A peptide comprising an N-terminal GSG linker is provided. In some embodiments, the GSG-T2A sequence comprises, consists essentially of, or consists of SEQ ID NO: 82. In some embodiments, the GSG-P2A sequence comprises, consists essentially of, or consists of SEQ ID NO:74.

[0216] In certain embodiments, the encoded binding protein comprises a TCR alpha chain and a TCR beta chain, and the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding the TCR alpha chain and the polynucleotide encoding the TCR beta chain. In further embodiments, the polynucleotide comprises an in-frame operably linked: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα), (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα), (iii) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-( pnTCRα)-(pnSCP3)-(pnTCRβ), (iv)(pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ), (v)(pnTCRβ)-( pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β), (vi)(pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8β)-(pnSC (pnCD8α), (vii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β), and (viii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain and pnCD8β is a polynucleotide encoding a C pnTCRα is a polynucleotide encoding a polypeptide comprising the extracellular portion of the D8 co-receptor α chain, pnTCRβ is a polynucleotide encoding a TCR α chain, and pnSCP1, pnSCP2, and pnSCP3 are each independently polynucleotides encoding a self-cleaving peptide, and these polynucleotides and / or the encoded self-cleaving peptides are the same or different, as appropriate.In some embodiments, SCP1 comprises SEQ ID NO:82, SCP2 comprises SEQ ID NO:74, and SCP3 comprises SEQ ID NO:74.

[0217] Additionally or alternatively, the polynucleotide encoding the binding protein can encode a furin cleavage site or other protease cleavage site located between two other polypeptides (e.g., between the TCR β chain and the TCR α chain).

[0218] In certain embodiments, an encoded polypeptide of the disclosure comprises one or more junction amino acids. A "junction amino acid" or "junction amino acid residue" refers to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, e.g., 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 encoding a fusion protein (e.g., amino acid residues resulting from the use of restriction enzyme sites during construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of a self-cleaving peptide adjacent to one or more domains of an encoded binding protein of the disclosure (e.g., a P2A peptide positioned between the TCR α and TCR β chains, the self-cleavage of which can leave one or more junction amino acids in the α chain, the TCR β chain, or both).

[0219] In further embodiments, the binding proteins are expressed as part of an encoding transgene construct, and / or the host cells of the present disclosure may encode one or more additional accessory proteins, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor beta chain; a CD8 co-receptor alpha chain, or both; or any combination thereof. Polynucleotides and transgene constructs and accessory components useful for encoding and expressing binding proteins (e.g., one or more of a safety switch protein, a selection marker, a CD8 co-receptor beta chain, or a CD8 co-receptor alpha chain) are described in PCT application PCT / US2017 / 053112, which polynucleotides, transgene constructs, and accessory components, including nucleotide and amino acid sequences, are hereby incorporated by reference. It will be understood that any or all of the binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor beta chains, or CD8 co-receptor alpha chains of the present disclosure 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.

[0220] Exemplary safety switch proteins include, for example, a pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux), which lacks an extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retains its native amino acid sequence and has type I transmembrane cell surface localization; tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011); caspase polypeptides (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.2016.07.011), 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. Sci. USA 105:623-628, 2008), and a truncated EGF receptor polypeptide (huEGFRt) with a conformationally intact binding epitope for a marker / safety switch polypeptide such as RQR (CD20+CD34; Philip et al., 2014).

[0221] Other accessory components useful for the modified host cells of the present disclosure include tags or selectable markers that allow for cell identification, sorting, isolation, enrichment, or tracking. For example, labeled host cells with desired traits (e.g., antigen-specific TCR and safety-switch protein) can be sorted from unlabeled cells in a sample and more efficiently activated and expanded for inclusion in a product of desired purity.

[0222] As used herein, the term "selection marker" includes a nucleic acid construct (and encoded gene product) that confers an identifiable change to a cell, allowing for the detection and positive selection of immune cells transduced with a polynucleotide containing the selection marker. RQR is a selection marker that contains a major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR includes a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is incorporated into the amino-terminal position of the 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 herein by reference). This construct allows for the selection of host cells expressing this construct using, for example, CD34-specific antibodies coupled to magnetic beads (Miltenyi), which allows for the selective deletion of T cells engineered to express the transgene using the clinically approved pharmaceutical antibody rituximab (Philip et al., 2014).

[0223] Further exemplary selection markers include several truncated type I transmembrane proteins not normally expressed on T cells: truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (see, e.g., Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Mavilio et al., Blood 83:1988-1997, 1994; Fehse et al., Mol. Ther. 1:448-456, 2000; each of which is incorporated herein in its entirety). A useful feature of CD19 and CD34 is the availability of a pre-made Miltenyi CliniMACs™ selection system that can target these markers for clinical grading. However, CD19 and CD34 are relatively large surface proteins that can burden the packaging capacity of vectors and the transcription efficiency of integrated vectors. Surface markers containing extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) can also be used. Any selectable marker can be used, provided it complies with Good Manufacturing Practice. In certain embodiments, a selectable marker is expressed along with a polynucleotide encoding a gene product of interest (e.g., a binding protein of the present disclosure, such as a TCR or CAR). Further examples of selectable markers include reporters, such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In certain embodiments, a selectable marker, such as CD34, can be expressed on cells and used to selectively enrich or isolate transduced cells of interest for use in the methods described herein (e.g., by immunomagnetic selection). As used herein, the CD34 marker is distinguished from an anti-CD34 antibody, or, for example, an scFv that binds CD34, a TCR, or another antigen recognition moiety.

[0224] In certain embodiments, the selectable marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

[0225] Regarding the RQR polypeptide, while not wishing to be bound by theory, it is believed that the distance from the host cell surface is important for the RQR polypeptide to function as a selection marker / safety switch (Philip et al., 2010 (supra)). In some embodiments, the encoded RQR polypeptide is contained within the beta chain, the alpha chain, or both, or a fragment or variant of either or both, of the encoded CD8 co-receptor. In specific embodiments, the modified host cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein comprising a beta chain containing an RQR polypeptide and further comprising a CD8 alpha chain.

[0226] In some embodiments, the encoded CD8 co-receptor comprises an alpha chain or a fragment or variant thereof. The amino acid sequence of the human CD8 co-receptor alpha chain precursor is known and is provided, for example, in UniProtKB-P30433 (see also UniProtKB-P31783; -P10732; and -P10731). In some embodiments, the encoded CD8 co-receptor comprises a beta chain or a fragment or variant thereof. The amino acid sequence of the human CD8 co-receptor beta chain precursor is known and is provided, for example, in UniProtKB-P10966 (see also UniProtKB-Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).

[0227] The isolated polynucleotides of the present disclosure may further comprise polynucleotides encoding a safety switch protein, a selectable marker, a CD8 co-receptor beta chain, or a CD8 co-receptor alpha chain disclosed herein, or may comprise polynucleotides encoding any combination thereof.

[0228] In any of the embodiments disclosed herein, the polynucleotide may 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, such as the GenScript® OptimumGene™ tool or GeneArt (Life Technologies). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more codons are optimized for expression in a host cell) and fully codon-optimized sequences. In embodiments in which the polynucleotide encodes more than one polypeptide (e.g., a TCR α chain, a TCR β chain, a CD8 co-receptor α chain, a CD8 co-receptor β chain, and one or more self-cleaving peptides), it will be understood that each polypeptide may be independently fully codon-optimized, partially codon-optimized, or not codon-optimized.

[0229] The amino acid and polynucleotide sequences for exemplary binding proteins "11N4A" and "11N6" are shown in Table 1. [Table 1]

[0230] Also provided is a polynucleotide comprising (i) an expression control sequence operably linked to (ii) a sequence encoding the amino acid sequence set forth in any one of SEQ ID NOs: 17, 27, 16, 26, 53, 43, 52, and 42. The expression control sequence can be heterologous to the sequence of (ii). The sequence of (ii) can be codon-optimized, for example, for expression in human T cells.

[0231] vector In another aspect, the disclosure provides an expression vector comprising any of the polynucleotides provided herein operably linked to an expression control sequence.

[0232] Also provided herein are vectors comprising the polynucleotides or transgene constructs of the present disclosure. Some examples of vectors include plasmids, viral vectors, cosmids, and the like. While 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), other vectors may integrate into the genome of the host cell upon introduction into the host cell or facilitate integration of a polynucleotide insert, thereby replicating along with the host genome (e.g., lentiviral vectors, retroviral vectors). In addition, some vectors are capable of directing the expression of genes to which they are operatively linked (such vectors are sometimes referred to as "expression vectors"). According to related embodiments, when one or more agents (e.g., polynucleotides encoding polypeptides as described herein) are co-administered to a subject, it is further understood that each agent may be present in a separate or the same vector, and that multiple vectors (each containing a different agent or the same agent) may be introduced into a cell or cell population or administered to a subject.

[0233] In certain embodiments, polynucleotides of the present disclosure may be operably linked to certain elements of a vector. For example, polynucleotide sequences necessary for effecting expression and processing of the coding sequence to which they are ligated may be operably 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 (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operably linked if they are adjacent to a gene of interest and expression control sequences that act in trans or remotely to control the gene of interest.

[0234] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from a lentiviral vector or a gamma-retroviral vector). Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), and paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma, mammalian type C, type B, type D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0235] A "retrovirus" is a virus with an RNA genome, which is reverse transcribed into DNA using reverse transcriptase, and the reverse-transcribed DNA is then integrated into the host cell genome. "Gammaretrovirus" refers to a genus of the Retroviridae family. Examples of gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. "Lentiviral vector," as used herein, refers to an HIV-based lentiviral vector for gene delivery, which can be integrative or non-integrative, has a relatively large packaging capacity, and is capable of transducing a range of different cell types. Lentiviral vectors are typically generated after transient transfection of three or more (packaging, envelope, and transfer) plasmids into producer cells. Like HIV, lentiviral vectors enter target cells through interaction of viral surface glycoproteins with receptors on the cell surface. Once inside, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is a substrate for viral integration into the DNA of infected cells. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. Self-inactivating lentiviral vectors can include, for example, a deletion of the 3' LTR of the viral genome that is transferred to the 5' LTR after one round of reverse transcription, and can include modifications to prevent transfer of enhancer and promoter elements in the 5' long terminal repeat (LTR) of the vector into transduced cells, resulting in a provirus that does not contain enhancer or promoter elements from the LTR. In some embodiments, the lentiviral vector is a third-generation lentiviral vector. Third-generation lentiviral vectors can utilize packaging systems split into two or more plasmids, for example, a plasmid encoding Rev and a plasmid encoding Gag and Pol.Third generation lentiviral vectors can utilize packaging systems that lack Tat or do not require Tat expression, and instead contain a chimeric 5' LTR fused to a heterologous promoter, for example, on a transfer plasmid.

[0236] In certain embodiments, the viral vector may be a gammaretrovirus, such as a Moloney murine leukemia virus (MLV)-derived vector. In other embodiments, the viral vector may be a more complex retrovirus-derived vector, such as a lentivirus-derived vector. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods for using retroviral and lentiviral viral vectors and packaging cells to transduce mammalian host cells with viral particles containing TCR or CAR transgenes are known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. For example, DNA viral vectors, including adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; other viral vectors, including vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-deficient HSV, and attenuated HSV, can also be used for polynucleotide delivery (Krisky et al., Gene Ther. 5:1517, 1998).

[0237] Other vectors developed for use in gene therapy can also be used with the compositions and methods of the present disclosure. Such vectors include those derived from baculovirus and alpha-virus (Jolly, DJ. 1999. Emerging Viral Vectors. pp 209-40 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).

[0238] When the viral vector genome contains multiple polynucleotides that are expressed as separate transcripts in a host cell, the viral vector may also contain additional sequences between the two (or more) transcripts to enable bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0239] In certain embodiments, the vector is capable of delivering a polynucleotide or transgene construct to a host cell (e.g., a hematopoietic progenitor cell or a cell of the human immune system). In specific embodiments, the vector delivers the polynucleotide or transgene construct to, for example, a CD4 + T cells, CD8 + T cells, CD4 - CD8 -The vector can be delivered to cells of the human immune system, such as double-negative T cells, stem cell memory T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof. In further embodiments, the vector can deliver the transgene construct to naive T cells, central memory T cells, effector memory T cells, or any combination thereof. In some embodiments, the vector encoding the polynucleotide or transgene construct of the present disclosure can further comprise a polynucleotide encoding 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 a portion thereof, to a host cell in gene therapy replacement or gene repair therapy. Alternatively, the nuclease used for chromosomal knockout or gene replacement or gene repair therapy can be delivered to a host cell independently of the vector encoding the polynucleotide or transgene construct of the present disclosure.

[0240] 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 cell of the human immune system. 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 γδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof. In still further embodiments, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0241] In any of the embodiments disclosed herein, the vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector or a gamma-retroviral vector.

[0242] Examples of transposon-based systems that can be used include, but are not limited to, sleeping beauty (e.g., derived from the salmonid genome), piggyback (e.g., derived from lepidopteran cells and / or the little brown bat (Myotis lucifugus)), mariner (e.g., derived from Drosophila), frog prince (e.g., derived from leopard frog), Tol2 (e.g., derived from medaka), and spinON.

[0243] host cell Also provided herein are host cells that encode and / or express the binding proteins (and optionally one or more accessory proteins provided herein, e.g., transduction markers, CD8 co-receptor polypeptides, etc.). In certain embodiments, host cells modified to contain the polynucleotides and / or expression vectors of the present disclosure and / or to express the binding proteins of the present disclosure are provided.

[0244] For example, any suitable host cell comprising an immune cell, e.g., a T cell, an NK cell, or an NK-T cell, modified to contain a heterologous polynucleotide can be modified to contain a heterologous polynucleotide encoding a binding protein of the present disclosure. In some embodiments, the modified immune cell is a CD4 + T cells, CD8 +Methods for transfecting / transducing T cells with a desired nucleic acid have been described (e.g., U.S. Patent Application Publication No. US2004 / 0087025), including adoptive transfer procedures using T cells of a 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. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US2011 / 0243972; US2011 / 0189141; Leen et al., Blood 109:2331, 2007). al., Ann. Rev. Immunol. 25:243, 2007), whereby it is contemplated to adapt these methodologies to embodiments of the present disclosure based on the teachings herein.

[0245] Any suitable method can be used to transfect or transduce cells, e.g., T cells, or to administer the polynucleotides or compositions of the methods of the present invention. Known methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercially available products, such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, biolistics, transposon-mediated transfer, and the like. Still further methods of transfecting or transducing host cells use vectors, which are described in more detail herein.

[0246] In certain embodiments, the host cells or modified cells comprise hematopoietic progenitor cells, stem cells (e.g., iPSCs), and / or human immune cells. In some embodiments, the immune cells comprise T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In further embodiments, the immune cells comprise CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, or any combination thereof. In certain further embodiments, the immune cells comprise CD4+ T cells and CD8+ T cells. In certain yet further embodiments, the CD4+ T cells, the CD8+ T cells, or both comprise (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain, where optionally the encoded polypeptide is or comprises the CD8 co-receptor alpha chain, (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain, where optionally the encoded polypeptide is or comprises the CD8 co-receptor beta chain, or (iii) the polynucleotide of (i) and the polynucleotide of (ii).

[0247] The host cell can be a peripheral blood mononuclear cell (PBMC). The host cell can be a lymphoid cell. The host cell can be a lymphocyte. The host cell can be a T cell. The host cell can be an alpha beta T cell (whether or not expressing an endogenous alpha-beta TCR). The host cell can be a gamma delta T cell (whether or not expressing an endogenous gamma-delta TCR). The host cell can be a B cell. The host cell can be a natural killer (NK) cell. The host cell can be a natural killer T (NKT) cell. The host cell can be a mammalian cell. The host cell can be a human cell.

[0248] The host cell can be a primary cell. The host cell can be an immortalized cell. The host cell can be a cell line. The host cell can be differentiated from a stem cell, such as an induced pluripotent stem cell (iPSC), an embryonic stem cell, a hematopoietic stem cell (HSC), etc.

[0249] In any of the above-described embodiments, host cells (e.g., immune cells) may be modified to reduce or eliminate expression of one or more endogenous genes encoding polypeptides involved in immune signaling or other related activities. Exemplary gene knockouts include those encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA molecules, TCR molecules, etc. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may be recognized as foreign by the allogeneic host receiving the modified immune cells, which may result in elimination of the modified immune cells (e.g., HLA alleles), 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., the endogenous TCR of the modified T cells, which binds to non-Ras antigens, thereby preventing the modified immune cells from binding to cells expressing Ras antigens).

[0250] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of the modified cells in autologous or allogeneic host settings, and may allow for universal administration of the cells (e.g., to any recipient regardless of HLA type). In certain embodiments, the modified cells are donor cells (e.g., allogeneic) or autologous cells. In certain embodiments, the modified cells of the present disclosure comprise a chromosomal gene knockout of one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA component (e.g., a gene encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), or a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(24):5697 (2012); and Torikai et al., Blood 122(8):1341 (2013); the gene editing techniques, compositions, and adoptive cell therapy methods of which are incorporated herein by reference in their entireties).

[0251] As used herein, the term "chromosomal gene knockout" refers to a genetic alteration or inhibitory agent introduced into a host cell that prevents (e.g., reduces, delays, suppresses, or prevents) the production of a functionally active endogenous polypeptide product by the host cell. Alterations that result in a chromosomal gene knockout can include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0252] In certain embodiments, chromosomal gene knockout or gene knockin is achieved by chromosome editing of a host cell. Chromosomal editing can be performed, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme capable of catalytically cleaving phosphodiester bonds within a polynucleotide chain. In certain embodiments, the endonuclease is capable of cleaving a target gene, thereby inactivating or "knocking out" the target gene. The endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease. Nucleic acid strand breaks caused by endonucleases are generally repaired by different mechanisms: homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule can be used for donor gene "knock-in," for target gene "knock-out," and, if necessary, to inactivate the target gene via 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 cut site (e.g., substitution, deletion, or addition of at least one nucleotide). NHEJ can be used to "knock out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0253] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein containing a zinc finger DNA-binding domain fused to a nonspecific DNA-cleavage domain, such as a Fokl endonuclease. Each zinc finger motif of approximately 30 amino acids binds approximately three base pairs of DNA, and amino acids at certain residues can be altered 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 specificities for desired DNA sequences, such as regions ranging in length from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and targeted integration of transgenes containing flanking sequences homologous to the genome at the DSB site is promoted by homology-directed repair. Alternatively, DSBs generated by ZFNs can result in knockout of the target gene through repair by non-homologous end joining (NHEJ). This is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the break site. In certain embodiments, gene knockout includes insertion, deletion, mutation, or a combination thereof, performed using ZFN molecules.

[0254] As used herein, "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 of which has a highly conserved sequence of 33-35 amino acids, typically diverging at the 12th and 13th amino acids. The TALE repeat domain is responsible for binding of the TALE to the target DNA sequence. These divergent amino acid residues are called repeat variable dinucleotides (RVDs) and are associated with specific nucleotide recognition. The natural (standard) code for DNA recognition of such TALEs has been determined as follows: the HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE results in TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) binds to an A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide. Non-standard (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. 2011 / 0301073, the atypical RVDs of which are incorporated herein by reference in their entirety). TALENs can be used to direct site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break with little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology-directed repair can introduce a transgene into the DSB site if homologous flanking sequences are present in the transgene. In certain embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, performed using a TALEN molecule.

[0255] As used herein, a "clustered regularly interspaced short palindromic repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that uses a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites (known as protospacers) within the genome by base-pair complementarity and cleave DNA if a short, conserved protospacer-associated motif (PAM) follows immediately 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 nuclease. Type I and Type III crRNA-guided surveillance complexes require multiple Cas subunits. The most studied Type II system contains at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA interact with the Cas9 nuclease, forming a duplex that can guide the Cas9 / crRNA:tracrRNA complex to a specific site in the target DNA through Watson-Crick base pairing between the spacer of the crRNA and the protospacer of the target DNA upstream of the PAM. The Cas9 nuclease cleaves a double-stranded break within the region defined by the crRNA spacer. Repair by NHEJ results in an insertion and / or deletion that disrupts expression of the target locus. Alternatively, a transgene with homologous flanking sequences can be introduced into the DSB site by 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).Additionally, the region of the guide RNA complementary to the target site can be altered or programmed to target a desired sequence (Xie et al., PLOS One 9:e100448, 2014; U.S. Patent Application Publication Nos. US2014 / 0068797, 2014 / 0186843, U.S. Patent No. 8,697,359, and PCT Publication No. WO2015 / 071474; each of which is incorporated by reference). In certain embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, made using a CRISPR / Cas nuclease system.

[0256] Exemplary gRNA sequences and methods for using them to knock out endogenous genes encoding immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNA, vectors, and gene knockout techniques of which are incorporated herein by reference in their entirety.

[0257] As used herein, "meganuclease," also known as "homing endonuclease," refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structural motifs: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII, and their recognition sequences are known (e.g., U.S. Pat. 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; Perler et al., Nucleic Acids Res. 22:1125-1127, 1994; Jasin, Trends Genet. 12:224-228, 1996; Gimble et al., J. Mol. Biol. 263:163-180, 1996; Argast et al., J. Mol. Biol. 280:345-353, 1998).

[0258] In certain embodiments, naturally occurring meganucleases can be used to facilitate site-specific genomic modification of targets selected from genes encoding PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, HLA, or genes encoding TCR components. In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 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 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Genetics 10:101-102, 2006). (See, e.g., Ther. 7:49-66, 2007; U.S. Patent Publication Nos. US2007 / 0117128, US2006 / 0206949, US2006 / 0153826, US2006 / 0078552, and US2004 / 0002092.) In a further embodiment, chromosomal gene knockouts are generated using homing endonucleases that have been modified with the modular DNA-binding domain of TALENs to create fusion proteins known as megaTALs. megaTALs can be used not only to knock out one or more target genes, but also to introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

[0259] In certain embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) that comprises 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 the encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, an HLA component, or a TCR component, or any combination thereof) in the host cell.

[0260] In certain embodiments, gene knockouts include insertions, deletions, mutations, or combinations thereof, and are made using a CRISPR / Cas nuclease system or a base editing system (Komor, AC, Kim, YB, Packer, MS, Zuris, JA, Liu, DR Nature 533, 420-424 (2016). Briefly, base editing is a genome editing approach that uses components from the CRISPR system, along with other enzymes, to introduce point mutations directly into cellular DNA or RNA without making double-stranded DNA breaks. Certain DNA base editors comprise a catalytically ineffective nuclease fused to a nucleobase deaminase enzyme, and in some cases, a DNA glycosylase inhibitor. RNA base editors function similarly, using RNA-targeting components. Base editors directly convert one base or base pair to another, allowing for the efficient incorporation of point mutations in non-dividing cells without producing excessive, undesired editing by-products. See, e.g., Rees H et al. Nature Reviews See Genetics (2018).

[0261] Chromosomal gene knockout can be confirmed directly by sequencing the DNA of the host immune cells after the knockout procedure or use of an agent. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., the absence of mRNA or polypeptide product encoded by the gene) after the knockout.

[0262] In certain embodiments, the chromosomal gene knockout comprises knockout of an HLA component gene selected from the α1 macroglobulin gene, the α2 macroglobulin gene, the α3 macroglobulin gene, the β1 microglobulin gene, or the β2 microglobulin gene.

[0263] In certain embodiments, the chromosomal gene knockout comprises knockout of a TCR component gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or a combination thereof.

[0264] 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 ... The host cell can comprise a binding protein (e.g., a TCR comprising the Va and Vβ regions and / or CDRs disclosed herein) that binds to a target antigen (e.g., a KRAS G12 mutant peptide, such as a KRAS G12V mutant peptide present in a peptide:HLA complex). Increased avidity can be determined, for example, by assays to determine expression of activation markers (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-a, IL-12, cytokines, interleukins, interferons) upon exposure to target cells expressing or presenting the target antigen, and / or by assays to determine the EC50 (e.g., the peptide dose that achieves half-maximal activation of a T cell population). In some embodiments, the host cells and control cells are both T cells, and the host cell and control cell populations can comprise the same, approximately the same, or substantially the same composition or amount(s) of T cell type(s) (e.g., CD4+, CD8+, or both).

[0265] 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 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 250-fold, or at least 1000-fold increased killing of target cells compared to a population of control cells (e.g., cells expressing a control binding protein specific for the same target antigen). Killing of target cells can be determined, for example, by in vitro cytotoxicity assays, at effector to target ratios of, for example, about 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. In some embodiments, the host cells and 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).

[0266] 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 14-fold, at least 16-fold, at least 18-fold, at least 19-fold, at least 20-fold, 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 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 25-fold, at least 26-fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at All of the antibodies exhibit a 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 increase in activation. Activation can be determined, for example, by assays to determine expression of activation markers (e.g., CD137, CD69, Granzyme B, CD107a, IFN-gamma, TNF-α, IL-12, cytokines, interleukins, interferons) upon exposure to target cells expressing or presenting the target antigen. 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, approximately the same, or substantially the same composition or amount(s) of T cell type(s) (e.g., CD4+, CD8+, or both).

[0267] In some embodiments, populations of host cells comprising a binding protein disclosed herein are resistant to depletion, e.g., exhibit effective tumor cell killing upon multiple re-challenges in vitro (e.g., at least 50 hours, at least 100 hours, at least 150 hours, at least 200 hours, or at least 250 hours, optionally with one or more re-challenges), or exhibit sustained control of tumor growth in vivo.

[0268] In some embodiments, a population of host cells comprising a binding protein disclosed herein is resistant to depletion compared to a population of control cells, e.g., exhibits superior tumor cell killing upon multiple re-challenges in vitro (e.g., for at least 50 hours, at least 100 hours, at least 150 hours, at least 200 hours, or at least 250 hours, optionally with one or more re-challenges), or exhibits superior control of tumor growth in vivo. 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).

[0269] Host Cell Compositions and Unit Doses In another aspect, provided herein are compositions and unit doses comprising the modified host cells of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0270] In certain embodiments, the host cell composition or unit dose comprises (i) at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4 + and (ii) at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8 T cells. +and a composition comprising T cells in a combination of about a 1:1 ratio, wherein the unit dose contains a reduced amount of or is substantially free of naive T cells (i.e., less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the population of naive T cells is present in the unit dose compared to a patient sample having a comparable number of PBMCs).

[0271] In some embodiments, the host cell composition or unit dose comprises (i) at least about 50% modified CD4 + a composition comprising T cells; and (ii) at least about 50% modified CD8 + In a further embodiment, the host cell composition or unit dose comprises (i) at least about 60% of the modified CD4 T cells and (ii) a composition comprising the modified CD4 T cells in a combination of about 1:1 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. + a composition comprising T cells; and (ii) at least about 60% modified CD8 + In yet a further embodiment, the host cell composition or unit dose comprises (i) at least about 70% engineered CD4 T cells and (ii) a composition comprising engineered CD4 T cells in a combination of about 1:1 ratio, wherein the unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. + a composition comprising T cells; and (ii) at least about 70% engineered CD8 + In some embodiments, the host cell composition or unit dose comprises (i) at least about 80% modified CD4 T cells and (ii) a composition comprising at least about 80% modified CD4 T cells in a combination of about 1:1 ratio, wherein the unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. + a composition comprising T cells; and (ii) at least about 80% modified CD8 + and a composition comprising T cells in a combination of about a 1:1 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 85% modified CD4 +a composition comprising T cells; and (ii) at least about 85% modified CD8 + and a composition comprising T cells in a combination of about a 1:1 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. In some embodiments, the host cell composition or unit dose comprises (i) at least about 90% modified CD4 + a composition comprising T cells; and (ii) at least about 90% modified CD8 + and a composition comprising T cells in a combination of about a 1:1 ratio, wherein the host cell composition or unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells.

[0272] In some embodiments, the composition comprises (i) a CD4+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells. In some embodiments, the composition further comprises (ii) a CD8+ cell population comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells.

[0273] In some embodiments, the host cell composition or unit dose comprises about a 1:1 ratio, about a 1:2 ratio, about a 1:3 ratio, about a 1:4 ratio, about a 1:5 ratio, about a 1:6 ratio, about a 1:7 ratio, about a 1:8 ratio, about a 1:9 ratio, about a 1:10 ratio, about a 2:1 ratio, about a 3:1 ratio, about a 4:1 ratio, about a 5:1 ratio, about a 6:1 ratio, about a 7:1 ratio, about a 8:1 ratio, about a 9:1 ratio, about a 10:1 ratio, about a 3:2 ratio, or about a 2:3 ratio of CD4+ to CD8+ T cells (e.g., CD4+ T cells modified to contain or express a binding protein disclosed herein to CD8+ T cells modified to contain or express a binding protein disclosed herein).

[0274] In some embodiments, the host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is at least 1:1, at least 1:2, at least 1:3, at least 1:4, at least 1:5, at least 1:6, at least 1:7, at least 1:8, at least 1:9, at least 1:10, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 3:2, or at least 2:3.

[0275] In some embodiments, the host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells that is at most 1:1, at most 1:2, at most 1:3, at most 1:4, at most 1:5, at most 1:6, at most 1:7, at most 1:8, at most 1:9, at most 1:10, at most 2:1, at most 3:1, at most 4:1, at most 5:1, at most 6:1, at most 7:1, at most 8:1, at most 9:1, at most 10:1, at most 3:2, or at most 2:3.

[0276] In some embodiments, the host cell composition or unit dose is about 1:10 to 10:1, 1:10 to 8:1, 1:10 to 7:1, 1:10 to 6:1, 1:10 to 5:1, 1:10 to 4:1, 1:10 to 3:1, 1:10 to 2:1, 1:10 to 1:1, 1:10 to 1:2, 1:10 to 1:3, 1:10 to 1:4, 1:10 to 1:5, 1:10 to 1:7, 1: 5~10:1, 1:5~8:1, 1:5~7:1, 1:5~6:1, 1:5~5:1, 1:5~4:1, 1:5~3:1, 1:5~2:1, 1:5~1:1, 1:5~1:2, 1:5~1:3, 1:5~1:4, 1:3~10:1, 1:3~8:1, 1:3~7:1, 1:3~6:1, 1:3~5:1, 1:3~4:1, 1:3~3:1, 1:3 ~2:1, 1:3~1:1, 1:3~1:2, 1:2~10:1, 1:2~8:1, 1:2~7:1, 1:2~6:1, 1:2~5:1, 1:2~4:1, 1:2~3:1, 1:2~2:1, 1:2~1:1, 1:1~10:1, 1:1~8:1, 1:1~7:1, 1:1~6:1, 1:1~5:1, 1:1~4:1, 1:1~3:1, 1:1~ Ratios of CD4+ to CD8+ T cells include 2:1, 2:1 to 10:1, 2:1 to 8:1, 2:1 to 7:1, 2:1 to 6:1, 2:1 to 5:1, 2:1 to 4:1, 2:1 to 3:1, 3:1 to 10:1, 3:1 to 8:1, 3:1 to 7:1, 3:1 to 6:1, 3:1 to 5:1, 3:1 to 4:1, 5:1 to 10:1, 5:1 to 8:1, 5:1 to 7:1, or 5:1 to 6:1.

[0277] The CD4+ T cells in the composition, host cell composition, or unit dose can be CD4+ T cells that have been modified or engineered to express a CD8 co-receptor as disclosed herein, e.g., using a vector or polynucleotide as disclosed herein.

[0278] It will be understood that the host cell compositions or unit doses of the present disclosure can comprise any host cell, or any combination of host cells, described herein. In certain embodiments, for example, the host cell compositions or unit doses comprise modified CD8+ T cells, modified CD4+ T cells, or both, which T cells are modified to encode a binding protein specific for the Ras peptide:HLA-A*11:01 complex. Additionally or alternatively, the host cell compositions or unit doses of the disclosure can include any host cell or combination of host cells described herein, and can be directed against different antigens (e.g., different Ras antigens, or different proteins or targets, e.g., BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., , MAGE-A1, MAGE-A3, and MAGE-A4), mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen-associated peptides bound to HLA, hTERT peptides bound to HLA, tyrosinase peptides bound to HLA, WT-1 peptides bound to HLA, LTβR, LIFRβ, LRP5, MUC1, OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, HA 1The unit dose may further comprise modified cells (e.g., immune cells, e.g., T cells) that express a binding protein specific for an antigen derived from Ras-HLA complexes (such as Ras-HLA, Robo1, alpha-fetoprotein (AFP), Frizzled, OX40, PRAME, and SSX-2). For example, the unit dose may comprise modified CD8 cells that express a binding protein that specifically binds to the Ras-HLA complex. + T cells and modified CD4 expressing a binding protein (e.g., CAR) that specifically binds to the PSMA antigen + T cells (and / or modified CD8 + T cells). It will also be understood that any of the host cells disclosed herein can be administered in combination therapy.

[0279] In any of the embodiments described herein, the host cell composition or unit dose contains an equal or approximately equal number of engineered CD45RA - CD3 + CD8 + and modified CD45RA - CD3 + CD4 + T M Contains cells.

[0280] In any of the embodiments described herein, the host cell composition or unit dose comprises one or more populations of cells (e.g., CD4+ or CD8+ cells) that have undergone CD62L-positive selection, e.g., to improve persistence in vitro.

[0281] Host cells may be engineered to contain or express the binding protein ex vivo, in vitro, or in vivo. In some embodiments, host cells are engineered ex vivo to express the binding protein. In some embodiments, host cells are engineered in vitro to express the binding protein. In some embodiments, host cells are engineered in vivo to express the binding protein.

[0282] use In an additional aspect, the present disclosure provides methods for treating or preventing the recurrence of a disease or disorder associated with a KRAS G12V, NRAS G12V, or HRAS G12V mutation in a subject, including, for example, cancers such as solid cancers and hematological malignancies. In certain exemplary embodiments, the disease or disorder is selected from the group consisting of pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; colon cancer; colorectal adenocarcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally a myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphocytic leukemia (ALL); cancer of the urinary tract; cancer of the small intestine; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma The diseases or disorders that can be treated by the compositions or methods disclosed herein include advanced or metastatic forms of the cancers disclosed herein.

[0283] "Treating" or "treatment" or "ameliorating" refers to the medical management of a disease, disorder, or condition in a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). Generally, an appropriate dose or treatment regimen comprising a composition of the disclosure (e.g., comprising a binding protein, polynucleotide, vector, host cell, host cell composition, unit dose, and / or immunogenic polypeptide) is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventative benefit includes improved clinical outcome; reduction or alleviation of symptoms associated with the disease; reduced occurrence of symptoms; improved quality of life; longer disease-free state; reduced extent of disease; stabilization of the disease state; delay in disease progression; remission; survival; long-term survival; or any combination thereof.

[0284] As used herein, a "therapeutically effective amount" or "effective amount" refers to an amount of a composition sufficient to produce a therapeutic effect such as improved clinical outcome, reduction or alleviation of symptoms associated with a disease, a reduction in the occurrence of symptoms, an improved quality of life, a longer disease-free state, a reduction in the extent of the disease, a stabilization of the disease state, a delay in disease progression, remission, survival, or prolonged survival in a statistically significant manner. When referring to an individual active ingredient administered alone or cells expressing a single active ingredient, the therapeutically effective amount refers to the effect of that ingredient alone or the cells expressing that ingredient. When referring to a combination, the therapeutically effective amount refers to the combined amount of the active ingredient or combined supplementary active ingredients and the cells expressing the active ingredient that produces a therapeutic effect, regardless of whether they are administered sequentially or simultaneously. The combination may also be cells expressing more than one active ingredient.

[0285] The terms "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refer to a biocompatible medium, e.g., physiological saline, that is suitable for administration to a human or other non-human mammalian subject and is generally recognized as safe or causing no serious adverse events, as described in more detail herein.

[0286] As used herein, "statistically significant" refers to a p-value of 0.050 or less, when calculated using a Student's t-test or other appropriate statistical test, indicating that the particular event or result being measured is unlikely to have occurred by chance.

[0287] Subjects that can be treated by the present invention are generally humans and other primate subjects, such as monkeys and apes for veterinary purposes. In any of the foregoing embodiments, the subject can be a human subject. The subject can be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. The subject can be a mammal. The compositions according to the present disclosure can be administered in a manner appropriate to the disease, condition, or disorder to be treated, as determined by one skilled in the medical arts. In any of the above embodiments, the modified host cells, host cell compositions, or unit doses as described herein are administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into lymph nodes, or into the cerebrospinal fluid to encounter target cells (e.g., leukemia cells). The appropriate dose, suitable duration, and frequency of administration of the composition will be determined by factors such as the patient's condition; the size, type, and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.

[0288] As used herein, the terms "adoptive immune therapy" or "adoptive immunotherapy" refer to the administration of naturally occurring or genetically engineered disease- or antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy can be autologous (the immune cells are derived from the recipient), allogeneic (the immune cells are derived from a donor of the same species who is not the recipient), or syngeneic (the immune cells are derived from a donor who is genetically identical or substantially identical to the recipient, e.g., an identical twin).

[0289] In some embodiments, the subject (eg, at least one cell in the subject) expresses a Ras antigen comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 2-3.

[0290] In some embodiments, the subject is HLA-A*11 + (e.g., HLA-A*11:01 + )

[0291] In certain embodiments, the method includes determining the subject's HLA type or types and / or identifying the presence of a Ras antigen prior to administering a therapy according to the present disclosure. In some embodiments, the subject's HLA type or types and / or the presence of a Ras antigen (e.g., a G12V mutation) have been determined prior to administering the treatment, e.g., the treatment is administered based at least in part on the HLA type(s) and / or the presence of a Ras antigen. In some embodiments, the method further includes genotyping the subject's tumor for the KRAS G12 allele prior to administration. In some cases, the subject is determined to have a KRAS G12V allele prior to administration.

[0292] The expression of HLA alleles can be determined, for example, by genetic sequencing (e.g., high-throughput next-generation sequencing (NGS)). This genetic determination of HLA expression is referred to herein as "HLA typing" and can be determined through a molecular approach in a clinical laboratory licensed for HLA typing. In some embodiments, HLA typing is performed using PCR amplification, followed by high-throughput NGS, and subsequent HLA determination. Herein, HLA haplotypes can be determined at major HLA loci (e.g., HLA-A, HLA-B, HLA-C, etc.).

[0293] HLA typing can be performed using any known method, including, for example, protein or nucleic acid testing. Examples of nucleic acid testing include sequence-based typing (SBT) and the use of sequence-specific oligonucleotide probes (SSOPs) or sequence-specific primers (SSPs). In certain embodiments, HLA typing is performed using PCR amplification followed by high-throughput next-generation sequencing (NGS) and subsequent HLA determination. In some embodiments, sequence typing is performed using a system available through Scisco Genetics (sciscogenetics.com / pages / technology.html, the contents of which are incorporated herein by reference in their entirety). Other methods for HLA typing include, for example, those disclosed in Mayor et al. PLoS One 10(5):e0127153 (2015), the methods and reagents of which are incorporated herein by reference.

[0294] In certain embodiments, the method comprises administering a composition comprising modified CD8+ and / or modified CD4+ T cells comprising a heterologous polynucleotide encoding a second binding protein as provided herein if the subject expresses HLA-A*11:01.

[0295] In the case of a host cell composition or unit dose, the amount of cells therein is at least one cell (e.g., one modified CD8 + T cell subpopulations (e.g., memory and / or naive CD8 as appropriate) + T cells); one modified CD4 + T cell subpopulations (e.g., memory and / or naive CD4 as appropriate) + T cells) or more typically 10 2 More than 10 cells, e.g., up to 10 4 , up to 10 5 , up to 10 6 , up to 10 7 , up to 10 8 , up to 10 9 , or 1010 In certain embodiments, the cells are greater than about 10 4 ~about 10 10 cells / m 2 in the range of, preferably, about 10 5 ~about 10 9 cells / m 2 In some embodiments, the administered dose is up to about 3.3 x 10 5 In some embodiments, the administered dose comprises up to about 1 x 10 cells / kg. 6 In some embodiments, the administered dose comprises up to about 3.3 x 10 cells / kg. 6 In some embodiments, the administered dose comprises up to about 1 x 10 cells / kg. 7 In certain embodiments, the modified immune cells comprise up to about 5 x 10 cells / kg. 4 cells / kg, 5×10 5 cells / kg, 5×10 6 cells / kg, or up to approximately 5 x 10 7 In certain embodiments, the modified immune cells are administered to a subject at a dose comprising at least about 5 x 10 cells / kg. 4 cells / kg, 5×10 5 cells / kg, 5×10 6 cells / kg, or up to approximately 5 x 10 7 The composition is administered to a subject in a dose containing cells / kg. The number of cells depends on the intended end use of the composition and the type of cells contained therein. For example, cells modified to contain a binding protein can include a cell population containing at least 30%, at least 35%, at least 40%, at least 45%, 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%, at least 95%, or more of such cells. For the uses provided herein, the cells are generally in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In embodiments, the desired cell density is typically 10 4cells / ml, generally greater than 10 7 cells / ml, generally greater than 10 8 cells / ml or greater. Cells may be administered as a single infusion or in multiple infusions over a range of time. Clinically relevant numbers of immune cells can be administered in a cumulative dose of 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , or 10 11 The unit dose of modified immune cells may be distributed over multiple infusions of equal to or more than 100 cells. In certain embodiments, the unit dose of modified immune cells may be co-administered (e.g., simultaneously or contemporaneously) with hematopoietic stem cells derived from an allogeneic donor. In some embodiments, one or more of the modified immune cells included in the unit dose are autologous to the subject.

[0296] In some embodiments, the unit dose or multiple unit doses comprise, consist essentially of, or consist of, e.g., at least 5x10^7, at least 1x10^8, at least 5x10^8, at least 1x10^9, at least 2.5x10^9, at least 5x10^9, at least 1x10^10, at least 1.5x10^10, at least 2x10^10, at least 3x10^10, at least 5x10^10, or at least 1x10^11 viable host cells encoding, containing, or expressing a binding protein disclosed herein.

[0297] In some embodiments, a unit dose or a plurality of unit doses comprises, consists essentially of, or consists of, e.g., at most 1x10^8, at most 5x10^9, at most 1x10^10, at most 1.5x10^10, at most 2x10^10, at most 2.5x10^10, at most 3x10^10, at most 4x10^10, at most 5x10^10, at most 1x10^11, at most 5x10^11, or at most 2x10^12 viable host cells encoding, containing, or expressing a binding protein disclosed herein.

[0298] In some embodiments, the unit dose or multiple unit doses may be, for example, about 1x10^8, about 5x10^8, about 1x10^9, about 2x10^9, about 3x10^9, about 4x10^9, about 5x10^9, about 6x10^9, about 7x10^9, about 8x10^9, about 9x10^9, about 1x10^10, about 1.1x10^10, about 1.2x10^10, about 1.3x10^10, about 1.4x10^10, about 1.5x10^10, about 1.6x10^10, about 1.7x10^10, about 1.8x10^10, about 1.9 ... x 10^10, about 1.3x10^10, about 1.4x10^10, about 1.5x10^10, about 1.6x10^10, about 1.7x10^10, about 1.8x10^10, about 1.9x10^10, about 2x10^10, about 3x10^10, about 4x10^10, about 5x10^10, about 7.5x10^10, about 10x10^10, or about 1x10^11 viable host cells.

[0299] In some embodiments, the unit dose or multiple unit doses may be, for example, about 1 x 10^8 to about 1 x 10^11, about 1 x 10^8 to about 5 x 10^10, about 1 x 10^8 to about 2 x 10^10, about 1 x 10^8 to about 1.5 x 10^10, about 1 x 10^8 to about 1 x 10^10, about 1 x 10^8 to about 5 x 10^9, about 1 x 10^9 to about 1 x 10^11, about 1 x 10^9 to about 5 x 10^10, about 1 x 10^9 to about 2 x 10^10, about 1 x 10^9 to about 1.5 x 10^10, about 1 x 10^9 to about 1 x 10^10, about 1 x 10 ^9 to approximately 5×10^9, approximately 5×10^9 to approximately 1×10^11, approximately 5×10^9 to approximately 5×10^10, approximately 5×10^9 to approximately 2×10^10, approximately 5×10^9 to approximately 1.5×10^10, approximately 5×10^9 to approximately 1×10^10, approximately 1×10^10 to approximately 1×10^11, approximately 1×10^10 to approximately 5×10^10 , about 1 x 10^10 to about 2 x 10^10, about 1 x 10^10 to about 1.5 x 10^10, about 1.5 x 10^10 to about 1 x 10^11, about 1.5 x 10^10 to about 5 x 10^10, or about 1.5 x 10^10 to about 2 x 10^10 viable host cells.

[0300] In some embodiments, the subject receiving the modified immune cells has previously received lymphodepleting chemotherapy, hi further embodiments, the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0301] In some embodiments, the method further comprises administering to the subject an inhibitor of an immune checkpoint molecule disclosed herein.

[0302] Pharmaceutical compositions (i.e., compositions) comprising the compositions disclosed herein (binding proteins, polynucleotides, vectors, host cells, host cell compositions, unit doses, and / or immunogenic polypeptides) and a pharmaceutically acceptable carrier, diluent, or excipient are also contemplated. Suitable excipients include water, saline, dextrose, glycerol, and the like, and combinations thereof. In embodiments, a composition comprising a fusion protein or host cell as disclosed herein further comprises a suitable infusion medium. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), 5% dextrose in water, or lactated Ringer's solution. The infusion medium can be supplemented with human serum albumin or other human serum components.

[0303] Pharmaceutical compositions can be administered in a manner appropriate to the disease or condition to be treated (or prevented), as determined by one skilled in the medical arts. The appropriate dose of the composition and a suitable duration and frequency of administration will be determined by factors such as the patient's health, the patient's size (i.e., weight, mass, or body area), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen will provide a sufficient amount of the composition(s) to provide a therapeutic and / or prophylactic benefit (such as those described herein, including improved clinical outcomes such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or reduced severity of symptoms).

[0304] An effective amount of a pharmaceutical composition refers to an amount sufficient to achieve the desired clinical results or beneficial treatment described herein, at the required dosage and for such period of time. An effective amount can be delivered in one or more administrations. When administration is to a subject already known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used to refer to treatment, while a "prophylactically effective amount" can be used to describe administering an effective amount to a subject who is susceptible to or at risk of developing a disease or disease state (e.g., recurrence) as a preventative measure.

[0305] The pharmaceutical compositions described herein can be provided in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain the stability of the formulation until infusion into a patient. While doses can vary, a preferred dose for administration of the modified immune cells described herein is about 10 4 cells / m 2 , about 5×10 4 cells / m 2 , about 10 5 cells / m 2 , about 5×10 5 cells / m 2 , about 10 6 cells / m 2 , about 5×10 6 cells / m 2 , about 10 7 cells / m 2 , about 5×10 7 cells / m 2 , about 10 8 cells / m 2 , about 5×10 8 cells / m 2 , about 10 9 cells / m 2 , about 5×10 9 cells / m 2 , about 10 10 cells / m 2 , about 5×10 10 cells / m 2 , or about 10 11 cells / m 2 In certain embodiments, the unit dose is about 10 4 cells / m2 ~about 10 11 cells / m 2 and developing suitable dosing and treatment regimens for using certain compositions described herein in a variety of treatment regimens, including, for example, parenteral or intravenous administration or formulations.

[0306] When the subject compositions are administered parenterally, they can also comprise sterile aqueous or oily solutions or suspensions. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol in a mixture with water. Aqueous solutions or suspensions may further contain one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts used. In addition, active compounds may be incorporated into sustained-release preparations and formulations. As used herein, dosage unit form refers to physically discrete units appropriate for single administration for a subject to be treated, each unit containing a predetermined amount of engineered immune cells or active compound calculated to produce the desired effect, together with an appropriate pharmaceutical carrier.

[0307] Generally, an appropriate dosage and treatment regimen provides a sufficient amount of active molecules or cells to provide benefit. Such a response can be monitored by establishing improved clinical outcomes (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects compared to untreated subjects. An increase in a pre-existing immune response to tumor proteins generally correlates with improved clinical outcomes. Such immune responses can generally be assessed using standard routine proliferation, cytotoxicity, or cytokine assays.

[0308] For prophylactic use, the dose should be sufficient to prevent, delay the onset of, or attenuate the severity of disease associated with the disease or disorder. The prophylactic benefit of the immunogenic compositions administered in accordance with the methods described herein can be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and analyzing the data obtained therefrom using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily performed by one of ordinary skill in the art.

[0309] As used herein, administering a composition refers to delivering it to a subject, regardless of the route or mode of delivery. Administration can be continuous or intermittent and parenteral. The composition can be administered locally (e.g., intratumorally) or systemically (e.g., intravenously). Administration can be for treating a subject already identified as having a recognized condition, disease, or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease, or disease state. Co-administration with adjunctive therapy can include simultaneous and / or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., modified immune cells with one or more cytokines; immunosuppressive therapeutic agents such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs, or any combination thereof).

[0310] In certain embodiments, multiple doses of the compositions described herein are administered to the subject, and the compositions may be administered to the subject at intervals of about 2 to about 4 weeks.

[0311] The therapeutic or preventive methods of the present disclosure can be administered to a subject as part of a course or regimen of treatment, and may include additional treatments before or after administration of the unit dose, cells, or compositions of the present disclosure. For example, in certain embodiments, the subject receiving the unit dose of modified immune cells is undergoing or has previously undergone hematopoietic cell transplantation (HCT; including myeloablative and non-myeloablative HCT). Techniques and regimens for performing HCT are known in the art and may include the transplantation of any suitable donor cells, such as cells derived from umbilical cord blood, bone marrow, or peripheral blood, hematopoietic stem cells, mobilized stem cells, or cells derived from amniotic fluid. Thus, in certain embodiments, one modified immune cell of the present disclosure can be administered together with or shortly after hematopoietic stem cells in a modified HCT regimen. In some embodiments, the HCT includes donor hematopoietic cells containing a chromosomal knockout of genes encoding HLA components, a chromosomal knockout of genes encoding TCR components, or both.

[0312] In further embodiments, the subject has previously received lymphodepleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, the lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0313] The methods according to the present disclosure may further comprise administering one or more additional agents to treat the disease or disorder in combination therapy. For example, in certain embodiments, the combination therapy comprises administering a composition of the present disclosure together (concurrently, simultaneously, or sequentially) with an immune checkpoint inhibitor. In some embodiments, the combination therapy comprises administering a composition of the present disclosure together with an agonist of a stimulatory immune checkpoint agent. In further embodiments, the combination therapy comprises administering a composition of the present disclosure together with a secondary therapy, such as a chemotherapeutic agent, radiation therapy, surgery, an antibody, or any combination thereof.

[0314] As used herein, the term "immune suppression agent" or "immunosuppression agent" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals to help control or suppress an immune response. For example, immunosuppressants include molecules that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immune suppression. Exemplary immunosuppressive agents that are targeted (e.g., with immune checkpoint inhibitors) include: PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244 / 2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

[0315] The immunosuppressant inhibitor (also called an immune checkpoint inhibitor) may be a chemical compound, an antibody, an antibody fragment, or a fusion polypeptide (e.g., an Fc fusion such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme, or an RNAi molecule, or a small organic molecule. In any of the embodiments disclosed herein, the method may include a composition of the present disclosure comprising one or more inhibitors of any one of the following immunosuppressive components, alone or in any combination:

[0316] In certain embodiments, compositions of the disclosure are used in combination with a PD-1 inhibitor, e.g., a PD-1-specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, or any combination thereof. In further embodiments, compositions of the disclosure are used in combination with a PD-L1-specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof. Also intended are cemiplimab; IBI-308; nivolumab + lilatolimab; BCD-100; camrelizumab; JS-001; spartalizumab; tislelizumab; AGEN-2034; BGBA-333 + tislelizumab; CBT-501; dostallimab; durvalumab + MEDI-0680; JNJ-3283; pazopanib hydrochloride + pembrolizumab; pidilizumab; REGN-1979 + cemiplimab; ABBV-181; ADUS-100 + spartalizumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; cemiplimab + REGN-3767; CS-1003; GLS-010; LZM-009; MEDI-5752; MGD-013; PF-06801591; Sym-021; Chi Slerizumab + pamiparib; XmAb-20717; AK-112; ALPN-202; AM-0001; antibodies antagonizing PD-1 for Alzheimer's disease; BH-2922; BH-2941; BH-2950; BH-2954; biologics antagonizing CTLA-4 and PD-1 for solid tumors; bispecifics targeting PD-1 and LAG-3 for oncology Monoclonal antibodies; BLSM-101; CB-201; CB-213; CBT-103; CBT-107; cellular immunotherapy + PD-1 inhibitors; CX-188; HAB-21; HEISCOIII-003; IKT-202; JTX-4014; MCLA-134; MD-402; mDX-400; MGD-019; monoclonal antibodies antagonizing PDCD1 in oncology;Monoclonal antibodies that antagonize PD-1 for oncology; oncolytic viruses that inhibit PD-1 for oncology; OT-2; PD-1 antagonist + PEG interferon alfa-2b; PEGMP-7; PRS-332; RXI-762; STIA-1110; TSR-075; vaccines targeting HER2 and PD-1 for oncology; vaccines targeting PD-1 for oncology and autoimmune disorders; XmAb-23104; antisense oligonucleotides that inhibit PD-1 for oncology; AT-16201; for oncology a bispecific monoclonal antibody that inhibits PD-1 for solid tumors; IMM-1802; a monoclonal antibody that antagonizes PD-1 and CTLA-4 for solid tumors and hematological tumors; a nivolumab biosimilar; a recombinant protein that agonizes CD278 and CD28 and antagonizes PD-1 for oncology; a recombinant protein that agonizes PD-1 for autoimmune and inflammatory disorders; SNA-01; SSI-361; YBL-006; AK-103; JY-034; AUR-012; BGB-108; PD-1 for solid tumors, Drugs inhibiting Gal-9 and TIM-3; ENUM-244C8; ENUM-388D4; MEDI-0680; monoclonal antibodies that antagonize PD-1 for metastatic melanoma and metastatic lung cancer; monoclonal antibodies that inhibit PD-1 for oncology; monoclonal antibodies targeting CTLA-4 and PD-1 for oncology; monoclonal antibodies that antagonize PD-1 for NSCLC; monoclonal antibodies that inhibit PD-1 and TIM-3 for oncology; monoclonal antibodies that inhibit PD-1 for oncology; hematology recombinant proteins that inhibit PD-1 and VEGF-A for clinical malignancies and solid tumors; small molecules that antagonize PD-1 for oncology; Sym-016; inebilizumab + MEDI-0680; vaccines targeting PDL-1 and IDO for metastatic melanoma; anti-PD-1 monoclonal antibodies plus cellular immunotherapy for glioblastoma; antibodies that antagonize PD-1 for oncology; monoclonal antibodies that inhibit PD-1 / PD-L1 for hematological malignancies and bacterial infections; monoclonal antibodies that inhibit PD-1 for HIV;or small molecules that inhibit PD-1 in solid tumors;

[0317] In certain embodiments, the compositions of the present disclosure are used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0318] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of CTLA4, hi certain embodiments, the compositions of the present disclosure are used in combination with a CTLA4-specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (e.g., abatacept, belatacept), or any combination thereof.

[0319] In certain embodiments, the compositions of the present disclosure are used in combination with a B7-H3-specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. The B7-H4 antibody binding fragment may be, for example, an scFv or fusion protein thereof, as described in Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO201640724A1 and WO2013 / 025779A1.

[0320] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of CD244.

[0321] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication No. WO2010 / 084158.

[0322] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of TIM3.

[0323] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of Gal9.

[0324] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of adenosine signaling, such as decoy adenosine receptors.

[0325] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of A2aR.

[0326] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of KIR, such as lirilumab (BMS-986015).

[0327] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0328] In certain embodiments, the compositions of the present disclosure are used in combination with an IDO inhibitor, such as levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-tirapazamine, or any combination thereof.

[0329] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof.

[0330] In certain embodiments, compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0331] In certain embodiments, compositions of the present disclosure are used in combination with an inhibitor of TIGIT, such as COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as COM701 (Compugen), or both.

[0332] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO2017 / 021526.

[0333] In certain embodiments, the compositions of the present disclosure are used in combination with a LAIR1 inhibitor.

[0334] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0335] In certain embodiments, the compositions of the present disclosure are used in combination with agents that increase the activity of (i.e., are agonists of) stimulatory immune checkpoint molecules. For example, the compositions of the present disclosure can be used in combination with the following: CD137 (4-1BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, CD27 agonists (e.g., CDX-1127), CD28 agonists (e.g., TGN1412, CD80, or CD86), CD40 agonists, or other agents that increase the activity of (i.e., are agonists of) stimulatory immune checkpoint molecules. agonists (e.g., CP-870,893, rhuCD40L, or SGN-40), CD122 agonists (e.g., IL-2), agonists of GITR (e.g., the humanized monoclonal antibodies described in PCT Patent Publication No. WO2016 / 054638), agonists of ICOS (CD278) (e.g., GSK3359609, mAb88.2, JTX-2011, Icos145-1, Icos314-8, or any combination thereof). In any of the embodiments disclosed herein, the method may comprise administering a composition of the present disclosure in conjunction with one or more agonists of stimulatory immune checkpoint molecules, including any of the foregoing, alone or in any combination.

[0336] In certain embodiments, the combination therapy comprises a composition of the present disclosure and a second-line therapy comprising one or more of an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflammatory solid tumor, radiation therapy, surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0337] In certain embodiments, the combination therapy method comprises administering the composition of the present disclosure and further administering radiation therapy or surgery.Radiation therapy is well known in the art and includes X-ray therapy, such as gamma irradiation, and radiopharmaceutical therapy.Surgery and surgical techniques suitable for treating a given cancer in a subject are well known to those skilled in the art.

[0338] In certain embodiments, the method of combination therapy comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent, including, but not limited to, inhibitors of chromatin function, topoisomerase inhibitors, anti-microtubule agents, DNA damaging agents, antimetabolites (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modifying analogs), DNA synthesis inhibitors, DNA interactors (such as intercalators), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer drugs such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine), and purine analogs, folate antagonists, and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disrupting agents such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine; epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, antiproliferative / antimitotic agents including cyclophosphamide, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin;enzymes (L-asparaginase, which metabolizes L-asparagine systemically and depletes cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), antiproliferative / antimitotic alkylating agents such as trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bica Anticoagulants (heparin, synthetic heparin salts, and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (vleverdin); immunosuppressants (cyclosporine, tacrolimus (FK-5 06), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin);mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11), and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.

[0339] Cytokines can be used to manipulate host immune responses toward anti-cancer activity. See, e.g., Floros & Tarhinis, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, used alone or in any combination with the compositions of the present disclosure.

[0340] Also provided herein is a method for modulating adoptive immunotherapy, the method comprising administering to a subject that has previously received modified host cells of the present disclosure comprising a heterologous polynucleotide encoding the safety switch protein, an amount of a cognate compound of a safety switch protein effective to eliminate previously administered modified host cells in the subject.

[0341] In certain embodiments, the safety switch protein comprises tEGFR and the cognate compound is cetuximab, or the safety switch protein comprises iCasp9 and the cognate compound is AP1903 (e.g., dimerized AP1903), or the safety switch protein comprises an RQR polypeptide and the cognate compound is rituximab, or the safety switch protein comprises a myc-binding domain and the cognate compound is an antibody specific for the myc-binding domain.

[0342] In yet a further aspect, methods are provided for producing compositions or unit doses of the present disclosure. In certain embodiments, the methods comprise combining (i) an aliquot of host cells transduced with a vector of the present disclosure with (ii) a pharmaceutically acceptable carrier. In certain embodiments, the vector of the present disclosure is used to transfect / transduce host cells (e.g., T cells) for use in adoptive transfer therapy (e.g., targeting a cancer antigen).

[0343] In some embodiments, the method further comprises culturing the transduced host cells and selecting for transduced cells that have integrated the vector (i.e., express the vector) prior to aliquoting. In further embodiments, the method comprises expanding the transduced host cells after culturing and selection and prior to aliquoting. In any embodiment of the method of the present invention, the produced composition or unit dose may be frozen or cryopreserved for later use. Any suitable host cells may be used to produce compositions or unit doses according to the method of the present invention, including, for example, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, or NK-T cells. In a specific embodiment, the method comprises: + T cells, CD4 + The present invention also includes host cells that are T cells, or both.

[0344] Also provided are any of the binding proteins, polynucleotides, expression vectors, host cells, host cell compositions, unit doses, and immunogenic polypeptides, taken alone or in any combination, for use in treating a disease or disorder associated with a KRAS G12D mutation or a KRAS G12V, or an NRAS G12D mutation or a NRAS G12V mutation, or an HRAS G12V mutation or a HRAS G12D mutation in a subject.

[0345] Also provided are any of the binding proteins, polynucleotides, expression vectors, host cells, host cell compositions, unit doses, and immunogenic polypeptides, taken alone or in any combination, for use in the manufacture of a medicament for treating a disease or disorder associated with a KRAS G12D mutation or a KRAS G12V, or an NRAS G12D mutation or a NRAS G12V mutation, or an HRAS G12V mutation or a HRAS G12D mutation in a subject.

[0346] In certain embodiments, the disease or disorder comprises cancer. In some embodiments, the cancer is a solid cancer or hematological malignancy. In certain embodiments, the disease or disorder comprises pancreatic cancer or carcinoma, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma, such as non-Hodgkin's lymphoma; chronic myelomonocytic leukemia; acute lymphocytic leukemia (ALL); cancer of the urinary tract; cancer of the small intestine; breast cancer or carcinoma; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic The tumor is selected from the group consisting of squamous cell neoplasm, neurofibromatosis type 1, rhabdomyosarcoma, soft tissue sarcoma, bladder cancer, sarcoma, glioblastoma, lung squamous cell carcinoma, anaplastic astrocytoma, chronic myeloid leukemia, diffuse large B-cell lymphoma, double-hit lymphoma, head and neck cancer, 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 cancer, refractory anemia with excess blasts-2, renal cell carcinoma, rhabdoid tumor, schwannoma, secondary AML, small cell lung cancer, therapy-related AML, thymic carcinoma, follicular thyroid carcinoma, malignant thyroid neoplasm, thyroid carcinoma, thyroid adenocarcinoma, urothelial carcinoma, or papillary thyroid carcinoma. In some embodiments, the method comprises parenteral or intravenous administration of the subject composition. In some embodiments, the methods include administering to the subject a binding protein, a polynucleotide, an expression vector, a host cell, a host cell composition, a unit dose, and / or multiple doses of an immunogenic polypeptide.

[0347] In certain embodiments, multiple doses are administered with an interval of about 2 to about 4 weeks between doses.

[0348] In certain embodiments, the composition comprises modified host cells. In some embodiments, the method comprises incubating the modified host cells for about 10 minutes. 4 cells / kg~about 10 11 cells / kg to the subject.

[0349] In certain embodiments, the method further comprises administering to the subject a cytokine, hi some embodiments, the cytokine comprises IL-2, IL-15, or IL-21.

[0350] In certain embodiments, the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

[0351] Also provided are methods that include introducing into a host (eg, T) cell a polynucleotide encoding a binding protein of the disclosure.

[0352] The present disclosure also provides the following non-limiting enumerated embodiments.

[0353] Embodiment 1. A binding protein comprising: (a) a T cell receptor (TCR) α chain variable (Vα) domain comprising the complementarity determining region 3 (CDR3α) amino acid sequence set forth in any one of SEQ ID NOs: 16, 17, 42, and 43, or a variant thereof having one, two, or three, optionally conservative amino acid substitutions; and / or (b) a TCR β chain variable (Vβ) domain comprising a CDR3 β amino acid sequence set forth in any one of SEQ ID NOs: 26, 27, 52, and 53, or a variant thereof having one, two, or three, optionally conservative amino acid substitutions; A binding protein capable of binding to a peptide:HLA complex, wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGAVGVGK (SEQ ID NO: 3), and the HLA comprises HLA-A*11.

[0354] Embodiment 2. The binding protein of embodiment 1, wherein the HLA comprises HLA-A*11:01.

[0355] Embodiment 3. The binding protein of embodiment 1 or 2, wherein the Vα domain and / or the Vβ domain is human, humanized, or chimeric, preferably human.

[0356] Embodiment 4. The binding protein according to any one of embodiments 1 to 3, comprising the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: (i) 17 and 27, respectively, or variants thereof, optionally with one, two or three conservative amino acid substitutions; (ii) 16 and 26, respectively, or variants thereof, optionally with one, two or three conservative amino acid substitutions; (iii) 53 and 43, respectively, or variants thereof, optionally with one, two or three conservative amino acid substitutions; or (iv) 52 and 42, respectively, or variants thereof, optionally with one, two or three conservative amino acid substitutions.

[0357] Embodiment 5. A binding protein according to any one of embodiments 1 to 4, comprising: (i) in the Vα domain, a CDR1α amino acid sequence as set forth in SEQ ID NO: 14 or 40, or a variant thereof, optionally with one or two conservative amino acid substitutions; (ii) in the Vα domain, a CDR2α amino acid sequence as set forth in SEQ ID NO: 15 or 41, or a variant thereof, optionally with one or two conservative amino acid substitutions; (iii) in the Vβ domain, a CDR1β amino acid sequence as set forth in SEQ ID NO: 24 or 50, or a variant thereof, optionally with one or two conservative amino acid substitutions; (iv) in the Vβ domain, a CDR2β amino acid sequence as set forth in SEQ ID NO: 25 or 51, or a variant thereof, optionally with one or two conservative amino acid substitutions; or (v) any combination of (i) to (iv).

[0358] Embodiment 6. The binding protein of any one of embodiments 1 to 5, comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 14, 15, 16 or 17, 24, 25, and 26 or 27, respectively.

[0359] Embodiment 7. The binding protein of any one of embodiments 1 to 5, comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 40, 41, 42 or 43, 50, 51, and 52 or 53, respectively.

[0360] Embodiment 8. (i) the Vα domain comprises, consists essentially of, or consists of an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 39; and / or (ii) The binding protein of any one of embodiments 1 to 7, wherein the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 23 or 49.

[0361] Embodiment 9. The binding protein of any one of embodiments 1-8, wherein the Vα domain comprises, consists essentially of, or consists of an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 13; and the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence having 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% identity to the...

Claims

**Claim 1** A binding protein, (a) a T cell receptor (TCR) α-chain variable (Vα) domain comprising a complementarity-determining region 3 (CDR3α) amino acid sequence set forth in any one of SEQ ID NOs: 16, 17, 42, and 43, or a variant thereof having 1, 2, or 3 conservative amino acid substitutions as necessary, and / or (b) a TCR β-chain variable (Vβ) domain comprising a CDR3β amino acid sequence set forth in any one of SEQ ID NOs: 26, 27, 52, and 53, or a variant thereof having 1, 2, or 3 conservative amino acid substitutions as necessary, wherein the binding protein is capable of binding to a peptide:HLA complex, the peptide comprising or consisting of the amino acid sequence VVVGAVGVGK (SEQ ID NO: 2) or VVGA VGVGK (SEQ ID NO: 3), and the HLA comprises HLA-A*11. **Claim 2** The binding protein according to claim 1, wherein the HLA comprises HLA-A*11:

01. **Claim 3** The binding protein according to claim 1 or 2, wherein the Vα domain and / or the Vβ domain is human, humanized, or chimeric, preferably human. **Claim 4** The binding protein according to any one of claims 1 to 3, comprising the CDR3α and CDR3β amino acid sequences set forth in SEQ ID NOs: (i) 17 and 27, respectively, or a variant thereof having 1, 2, or 3 conservative amino acid substitutions as necessary, (ii) 16 and 26, respectively, or a variant thereof having 1, 2, or 3 conservative amino acid substitutions as necessary, (iii) 53 and 43, respectively, or a variant thereof having 1, 2, or 3 conservative amino acid substitutions as necessary, or (iv) 52 and 42, respectively, or a variant thereof having 1, 2, or 3 conservative amino acid substitutions as necessary. **Claim 5** (i) In the Vα domain, the CDR1α amino acid sequence set forth in SEQ ID NO: 14 or 40, or a variant thereof having one or two conservative amino acid substitutions as necessary; (ii) In the Vα domain, the CDR2α amino acid sequence set forth in SEQ ID NO: 15 or 41, or a variant thereof having one or two conservative amino acid substitutions as necessary; (iii) In the Vβ domain, the CDR1β amino acid sequence set forth in SEQ ID NO: 24 or 50, or a variant thereof having one or two conservative amino acid substitutions as necessary; (iv) In the Vβ domain, the CDR2β amino acid sequence set forth in SEQ ID NO: 25 or 51, or a variant thereof having one or two conservative amino acid substitutions as necessary; or (v) The binding protein according to any one of claims 1 to 4, comprising any combination of (i) to (iv).

6. The binding protein according to any one of claims 1 to 5, comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NO: 14, 15, 16 or 17, 24, 25, and 26 or 27, respectively.

7. The binding protein according to any one of claims 1 to 5, comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NO: 40, 41, 42 or 43, 50, 51, and 52 or 53, respectively.

8. (i) The Vα domain comprises, or consists of, an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 13 or 39, and / or (ii) The binding protein according to any one of claims 1 to 7, wherein the Vβ domain comprises, or consists of, an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 23 or 49.

9. The binding protein according to any one of claims 1 to 8, wherein the Vα domain comprises, or consists of, an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain comprises, or consists of, an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO:

23.

10. The Vα domain comprises, or consists of, an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 39, and the Vβ domain comprises, or consists of, an amino acid sequence having 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% identity to the amino acid sequence set forth in SEQ ID NO: 49, the binding protein according to any one of claims 1 to 8.

11. The Vα domain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 13, and the Vβ domain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 23, the binding protein according to any one of claims 1 to 10.

12. The Vα domain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 39, and the Vβ domain comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 49, the binding protein according to any one of claims 1 to 10.

13. The binding protein according to any one of claims 1 to 12, further comprising a TCR α-chain constant domain (Cα) and / or a TCR β-chain constant domain (Cβ).

14. The Cα has 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 the amino acid sequence set forth in any one of SEQ ID NOs: 18, 19, 44, 45, and 69, or comprises, or consists of, an amino acid sequence comprising, or consisting of, the amino acid sequence, the binding protein according to claim 13.

15. The binding protein according to claim 13 or 14, wherein Cβ has 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 the amino acid sequence set forth in any one of SEQ ID NOs: 28, 29, 54, 55, and 70-73, or comprises the amino acid sequence, or comprises an amino acid sequence consisting of the amino acid sequence, or consists of the same.

16. wherein Cα and Cβ are SEQ ID NO (i) 18 and 28 respectively, (ii) 19 and 29 respectively (iii) 44 and 54 respectively, or (iv) 45 and 55 respectively, and has 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 the amino acid sequence set forth therein, or comprises the amino acid sequence, or comprises an amino acid sequence consisting of the amino acid sequence, or consists of the same, the binding protein according to any one of claims 13-15.

17. The binding protein according to any one of claims 13-16, wherein Cα, Cβ, or both comprise a modification(s) that promotes preferential pairing of Cα and Cβ.

18. The binding protein according to any one of claims 13-16, wherein Cα and Cβ each comprise an introduced cysteine residue that promotes preferential pairing of Cα and Cβ.

19. The binding protein according to any one of claims 13-16, wherein Cα comprises a T48C substitution and Cβ comprises an S57C substitution, which promotes preferential pairing of Cα and Cβ.

20. comprising a TCRα chain and a TCRβ chain, wherein the TCRα chain and the TCRβ chain are (i) 12 and 22 of SEQ ID NO respectively, (ii) 20 and 30 of SEQ ID NO respectively, (iii) 12 and 30 of SEQ ID NO respectively, (iv) 20 and 22 of SEQ ID NO respectively, (v) 38 and 48 of SEQ ID NO respectively, (vi) 46 and 56 of SEQ ID NO respectively, (vii) 38 and 56 of SEQ ID NO respectively, or (viii) 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 the amino acid sequences set forth in SEQ ID NOs: 46 and 48, or comprising the amino acid sequences, or consisting of the amino acid sequences, or consisting of an amino acid sequence comprising the amino acid sequences, the binding protein according to any one of claims 1 to 19.

21. The binding protein according to any one of claims 1 to 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).

22. The binding protein according to claim 21, wherein the binding protein comprises a TCR.

23. The binding protein according to any one of claims 1 to 22, wherein the binding protein has 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 the functional avidity for the peptide.

24. An isolated polynucleotide encoding the binding protein according to any one of claims 1 to 23.

25. having 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%, or at least 99% identity to the polynucleotide sequences set forth in any one of SEQ ID NOs: 5 to 10 and 33 to 36, or comprising the polynucleotide sequences, or consisting of the polynucleotide sequences, or consisting of a polynucleotide comprising the polynucleotide sequences, the polynucleotide according to claim 24.

26. (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain, wherein optionally the encoded polypeptide is the CD8 co-receptor alpha chain or comprises it, a polynucleotide (ii)A polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is the CD8 co-receptor β chain or comprises it, a polynucleotide, or (iii)The polynucleotide according to claim 24 or 25, further comprising the polynucleotide of (i) and the polynucleotide of (ii). **Claim 27** (a)The polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor α chain, and (b)The polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor β chain, and (c)A polynucleotide encoding a self-cleaving peptide, disposed between the polynucleotide of (a) and the polynucleotide of (b), the polynucleotide according to claim 26. **Claim 28** Encoding a self-cleaving peptide, and (1)Between the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor α chain, and / or (2)The polynucleotide according to claim 26 or 27, further comprising a polynucleotide disposed between the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor β chain. **Claim 29** Operably linked in-frame, (i)(pnCD8α)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnBP)、 (ii) (pnCD8β)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnBP), (iii) (pnBP)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnCD8β), (iv) (pnBP)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnCD8α), (v) (pnCD8α)-(pnSCP １ )-(pnBP)-(pnSCP ２ )-(pnCD8β), or (vi) (pnCD8β)-(pnSCP １ )-(pnBP)-(pnSCP ２ )-(pnCD8α), and pnCD8α is the polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor α chain, pnCD8β is the polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor α chain, pnBP is the polynucleotide encoding a binding protein, pnSCP １ and pnSCP ２ each independently is a polynucleotide encoding a self-cleaving peptide, and the polynucleotide and / or the encoded self-cleaving peptide are the same or different as necessary, the polynucleotide according to any one of claims 26 to 28. **Claim 30** The encoded binding protein comprises a TCRα chain and a TCRβ chain, and the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between a polynucleotide encoding a TCRα chain and a polynucleotide encoding a TCRβ chain, the polynucleotide according to any one of claims 26 to 29. **Claim 31** Operably linked in-frame, (i) (pnCD8α)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnTCRβ)-(pnSCP ３ )-(pnTCRα), (ii) (pnCD8β)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnTCRβ)-(pnSCP ３ )-(pnTCRα), (iii) (pnCD8α)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnTCRα)-(pnSCP ３ )-(pnTCRβ), (iv) (pnCD8β)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnTCRα)-(pnSCP ３ )-(pnTCRβ), (v)(pn TCRβ)-(pn SCP １ )-(pn TCRα)-(pn SCP ２ )-(pn CD8α)-(pn SCP ３ )-(pn CD8β), (vi) (pn TCRβ)-(pn SCP １ )-(pn TCRα)-(pn SCP ２ )-(pn CD8β)-(pn SCP ３ )-(pn CD8α), (vii) (pnTCRα)-(pnSCP １ )-(pnTCRβ)-(pnSCP ２ )-(pnCD8α)-(pnSCP ３ )-(pnCD8β), (viii) (pnTCRα)-(pnSCP １ )-(pnTCRβ)-(pnSCP ２ )-(pnCD8β)-(pnSCP ３ )-(pnCD8α), and pnCD8α is the polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor α chain, pnCD8β is the polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor α chain, pnTCRα is the polynucleotide encoding the TCRα chain, pnTCRβ is the polynucleotide encoding the TCRβ chain, pnSCP １ 、 pnSCP ２ 、 and pnSCP ３ are each independently a polynucleotide encoding a self-cleaving peptide, and the polynucleotide and / or the encoded self-cleaving peptide are the same or different as required, the polynucleotide according to claim 30.

32. The polynucleotide according to claim 31, wherein the pnSCP1 encodes a T2A peptide, the pnSCP2 encodes a P2A peptide, and the pnSCP3 encodes a P2A peptide.

33. The polynucleotide according to any one of claims 24 to 32, encoding 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 the amino acid sequence set forth in any one of SEQ ID NOs: 11, 21, 37, 47, 31, 32, 57, and 58, or comprising the amino acid sequence, or consisting of the amino acid sequence.

34. The polynucleotide according to claim 33, encoding (i) 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 the amino acid sequence set forth in SEQ ID NO: 11, or comprising the amino acid sequence, or consisting of the amino acid sequence, and (ii) 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 the amino acid sequence set forth in SEQ ID NO: 21, or comprising the amino acid sequence, or consisting of the amino acid sequence.

35. The polynucleotide according to claim 33, encoding 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 the amino acid sequence set forth in SEQ ID NO: 37, or comprising the foregoing amino acid sequence, or consisting of the foregoing amino acid sequence, and (ii) 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 the amino acid sequence set forth in SEQ ID NO: 47, or comprising the foregoing amino acid sequence, or consisting of the foregoing amino acid sequence.

36. The polynucleotide according to any one of claims 24 to 35, which is a polynucleotide sequence that is codon-optimized for expression in a host cell, or comprises the same, wherein, optionally, the host cell is a human immune system cell, and further optionally, is a T cell.

37. An expression vector comprising the polynucleotide according to any one of claims 24 to 36, operably linked to an expression control sequence.

38. The expression vector according to claim 37, wherein the expression control sequence comprises an MSCV promoter.

39. The expression vector according to claim 37 or 38, wherein the expression control sequence drives the expression of a single mRNA encoding the extracellular portion of the CD8 co-receptor α chain, the extracellular portion of the CD8 co-receptor β chain, the TCRα chain, and the TCRβ chain.

40. The expression vector according to any one of claims 37 to 39, wherein the vector is capable of delivering the polynucleotide to a host cell.

41. The expression vector according to claim 40, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

42. The human immune system cells are CD4 ＋ T cells, CD8 ＋ T cells, CD4 － CD8 － The expression vector according to claim 41, which is double-negative T cells, γδ T cells, natural killer cells, natural killer T cells, macrophages, monocytes, dendritic cells, or any combination thereof.

43. The expression vector according to claim 42, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

44. The expression vector according to any one of claims 37 to 43, wherein the vector is a viral vector.

45. The expression vector according to claim 44, wherein the viral vector is a lentiviral vector or a γ-retroviral vector.

46. The expression vector according to claim 44, wherein the viral vector is a self-inactivating lentiviral vector.

47. The expression vector according to claim 44 or 46, wherein the viral vector is a third-generation lentiviral vector.

48. A host cell modified to contain the polynucleotide according to any one of claims 24 to 36 and / or the expression vector according to any one of claims 37 to 47, and / or to express the binding protein according to any one of claims 1 to 23.

49. The host cell according to claim 48, wherein the modified cell contains hematopoietic progenitor cells and / or human immune cells.

50. The host cell according to claim 49, wherein the immune cells include T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof.

51. The immune cells are CD4 ＋ T cells, CD8 ＋ T cells, CD4 － CD8 － double-negative T cells, γδ T cells, naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof, Optionally, the immune cells include CD4 ＋ T cells and CD8 ＋ T cells, and optionally, the CD4 ＋ T cells, the CD8 ＋ T cells, or both, are (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 coreceptor alpha chain, optionally wherein the encoded polypeptide is the CD8 coreceptor alpha chain or comprises it, (ii) a polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 coreceptor beta chain, optionally wherein the encoded polypeptide is the CD8 coreceptor beta chain or comprises it, or (iii) a polynucleotide comprising the polynucleotide of (i) and the polynucleotide of (ii), the host cell according to claim 50.

52. The host cell according to any one of claims 48 to 51, wherein the modified cell contains chromosomal gene knockout of the PD-1 gene, LAG3 gene, TIM3 gene, CTLA4 gene, HLA component gene, TIGIT gene, TCR component gene, FasL gene, or any combination thereof.

53. The host cell according to claim 52, wherein the chromosomal gene knockout includes knockout of an HLA component gene selected from the α1 macroglobulin gene, α2 macroglobulin gene, α3 macroglobulin gene, β1 microglobulin gene, or β2 microglobulin gene.

54. The host cell according to claim 52 or 53, wherein the chromosomal gene knockout includes knockout of a TCR component gene selected from the TCRα variable region gene, TCRβ variable region gene, TCR constant region gene, or any combination thereof.

55. A composition comprising the host cell according to any one of claims 48 to 54 and a pharmaceutically acceptable carrier, diluent, or excipient.

56. (ii) at a ratio of about 1:1, at least about 30% of modified CD8 ＋ T cells in combination with a composition comprising at least about 30% of modified CD4 ＋ The composition according to claim 55, comprising T cells.

57. The composition according to claim 55 or 56, wherein the composition substantially does not contain naive T cells.

58. A composition comprising: (ix) the binding protein according to any one of claims 1 to 23, (x) A polynucleotide according to any one of claims 24 to 36, (xi) An expression vector according to any one of claims 37 to 47, and / or (xii) A host cell according to any one of claims 48 to 54, A composition comprising a pharmaceutically acceptable carrier, excipient, or diluent.

59. A method for treating a disease or disorder associated with a KRAS G12V mutation or an NRAS G12V mutation or an HRAS G12V mutation in a subject, said method comprising administering to said subject an effective amount of (i) A binding protein according to any one of claims 1 to 23, (ii) A polynucleotide according to any one of claims 24 to 36, (iii) An expression vector according to any one of claims 37 to 47, (iv) A host cell according to any one of claims 48 to 54, wherein optionally said host cell comprises CD8+ T cells, CD4+ T cells, or both, and wherein optionally said host cell is autologous, allogeneic, or syngeneic to said subject, and / or (v) A composition according to any one of claims 55 to 58.

60. The method according to claim 59, wherein said disease or disorder comprises cancer, and wherein said cancer is optionally a solid cancer or a hematological malignancy.

61. The method according to claim 59 or 60, wherein the disease or disorder is pancreatic cancer or tumor, optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or tumor; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer or tumor; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, optionally myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or tumor; melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; squamous cell head and neck cancer; hepatocellular cancer; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell cancer; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic cancer; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid gland cancer; urothelial cancer; colon cancer; colorectal adenocarcinoma; papillary thyroid cancer; or a progressive or metastatic form thereof.

62. The method according to any one of claims 59 to 61, wherein the binding protein, polynucleotide, vector, host cell, or composition is administered parenterally or intravenously to the subject.

63. The method according to any one of claims 59 to 62, wherein the method comprises administering to the subject one or more of the plurality of doses of any one of (i) to (v).

64. The method according to claim 63, wherein the plurality of doses are administered at an administration interval of about 2 to about 4 weeks.

65. The composition comprises the host cell or the composition comprising the host cell, and the method comprises administering to the subject the host cell or composition at a dose of about 10 ４ cells / kg to about 10 １１ cells / kg. The method according to any one of claims 59 to 64.

66. The method according to any one of claims 59 to 65, further comprising administering, if necessary, in a single dose, at least 5×10^8, at least 1×10^9, at least 5×10^9, at least 1×10^10, at least 1.5×10^10, at least 2×10^10, or at least 5×10^10 viable host cells comprising the binding protein to the subject.

67. The method according to any one of claims 59 to 65, further comprising administering, if necessary, in a single dose, up to 5×10^9, up to 1×10^10, up to 1.5×10^10, up to 2×10^10, up to 5×10^10, up to 1×10^11, or up to 5×10^11 viable host cells comprising the binding protein to the subject.

68. The method according to any one of claims 59 to 65, further comprising administering, if necessary, in a single dose, about 5×10^9, about 6×10^9, about 7×10^9, about 8×10^9, about 9×10^9, about 1×10^10, about 1.1×10^10, about 1.2×10^10, about 1.3×10^10, about 1.4×10^10, about 1.5×10^10, about 1.6×10^10, about 1.7×10^10, about 1.8×10^10, about 1.9×10^10, or about 2×10^10 viable host cells comprising the binding protein to the subject.

69. The method according to any one of claims 59 to 65, further comprising administering, if necessary, in a single dose, about 5×10^9 to about 1×10^11, about 5×10^9 to about 5×10^10, about 5×10^9 to about 2×10^10, about 5×10^9 to about 1.5×10^10, about 5×10^9 to about 1×10^10, about 1×10^10 to about 1×10^11, about 1×10^10 to about 5×10^10, about 1×10^10 to about 2×10^10, or about 1×10^10 to about 1.5×10^10 viable host cells comprising the binding protein to the subject.

70. The method according to any one of claims 59 to 69, further comprising determining that the subject expresses HLA-A*11, and optionally HLA-A*11:01, before administering the binding protein, polynucleotide, vector, host cell, or composition.

71. The method according to any one of claims 59 to 70, further comprising administering a cytokine to the subject. **Claim 72** The method according to claim 71, wherein the cytokine comprises IL-2, IL-15, or IL-21. **Claim 73** The method according to any one of claims 59 to 72, wherein the subject has received or is receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent. **Claim 74** A disease or disorder associated with a KRAS G12V or NRAS G12V or HRAS G12V mutation in a subject, wherein, optionally, the disease or disorder includes cancer, and further optionally, the cancer is a solid cancer or a hematological malignancy, and optionally, the disease or disorder is pancreatic cancer or carcinoma, and optionally, pancreatic ductal adenocarcinoma (PDAC); colorectal cancer or carcinoma; lung cancer, and optionally, non-small cell lung cancer; biliary tract cancer; endometrial cancer or carcinoma; cervical cancer; ovarian cancer; bladder cancer; liver cancer; myeloid leukemia, and optionally, myeloid leukemia such as acute myeloid leukemia; myelodysplastic syndrome; lymphoma such as non-Hodgkin lymphoma; chronic myelomonocytic leukemia; acute lymphoblastic leukemia (ALL); urinary tract cancer; small intestine cancer; breast cancer or carcinoma; melanoma (optionally, cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid cancer; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer; sarcoma; glioblastoma; squamous cell lung cancer; anaplastic astrocytoma; chronic myeloid leukemia; diffuse large B-cell lymphoma; double-hit lymphoma; head and neck cancer; head and neck squamous cell cancer; hepatocellular cancer; malignant peripheral nerve sheath tumor; mantle cell lymphoma; myelodysplastic / myeloproliferative neoplasm, unclassifiable; peripheral T-cell lymphoma; prostate cancer; refractory anemia with excess blasts-2; renal cell cancer; rhabdoid tumor; schwannoma; secondary AML; small cell lung cancer; therapy-related AML; thymic cancer; follicular thyroid cancer; malignant thyroid neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial cancer; colon cancer; colorectal adenocarcinoma; papillary thyroid cancer; or a method for treating a disease or disorder selected from their advanced or metastatic forms, for use in the use according to any one of claims 1 to 23 of the binding protein, any one of claims 24 to 36 of the polynucleotide, any one of claims 37 to 47 of the expression vector, any one of claims 48 to 54 of the host cell, wherein, optionally, the host cell includes CD8+ T cells, CD4+ T cells, or both, and / or any one of claims 55 to 58 of the composition.

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

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