Binding proteins specific for ras neoantigen and uses thereof

Abstract

The present disclosure provides compositions and methods for targeting Ras antigens, for example, to treat or prevent cancer. The disclosed embodiments include binding proteins, such as T cell receptors that bind to the Ras antigen: HLA complex. The disclosed binding proteins are highly sensitive to antigens, which binding proteins are capable of inducing activation of host T cells at low concentrations of peptide antigens. In certain embodiments, binding proteins of the present disclosure are non-alloreactive, substantially non-alloreactive, and / or alloreactivity with a lower risk against (i) amino acid sequences from human proteomes and / or (ii) 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 immunotherapy for the treatment of various cancers.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 342,025 filed on May 13, 2022, U.S. Provisional Patent Application No. 63 / 380,551 filed on October 21, 2022, and U.S. Provisional U.S. Patent Application No. 63 / 488,758 filed on March 6, 2023, the entire disclosures of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (502WO_SeqListing.xml; size: 189 kilobytes; and creation date: May 11, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Ras family proteins are small GTPases involved in intracellular signaling, including, for example, 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 disrupt negative growth signaling can lead to sustained cell proliferation. KRAS is one of the most commonly mutated proto-oncogenes in a variety of human cancers, including melanoma, endometrial cancer, thyroid cancer, pancreatic cancer, colorectal cancer, breast cancer, ovarian cancer, and lung cancer, as well as in some myeloid leukemias, such as AML. Pharmacological inhibitors targeting KRAS G12C have been developed, but cancers have been reported to have both intrinsic and adaptive resistance to these inhibitors (e.g., Awad et al. New England Journal of Medicine (NEJM) 384:2382-2393 (2021)). New therapies targeting mutant RAS proteins are needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figures 1A-1E This study involves identifying KRAS G12V-specific T cell receptors (TCRs) from the T cell repertoire of healthy human donors. Figure 1A (Left) Schematic diagram showing a method for identifying HLA-A11-restricted mutant KRAS (mKRAS)-specific T cell lines from donor samples, and (right) a method for producing TNFα by CD8+ T cells expressing an mKRAS-specific TCR in the absence (left) or presence (right) of an mKRAS G12V peptide. ( Figure 1B (Top) Schematic diagram of the method used to sort and sequence mKRAS-reactive CD8+ T cells and (bottom) engineer CD8+ T cells to heterologously express an mKRAS-specific TCR. Fifty-six mKRAS-specific TCRs (G12V-specific or G12D-specific) were isolated and assayed for sensitivity and cytotoxicity. ( Figure 1C ) In vitro enrichment fold of T cell clones in the presence and absence of KRAS G12V mutant peptide. ( Figure 1D ) In vitro activation of TCR-transduced T cells in the presence of varying concentrations of KRAS G12V mutant peptide, as assessed by the percentage of T cells expressing GFP under the control of the Nur77 locus. T cells were transduced to express TCRs as indicated in the figure legend. ( Figure 1E ) Log EC50 KRAS G12V 9-mer peptide value (represents the concentration of KRAS G12V peptide required for TCR-transduced T cells to generate a half-maximal response of Nur77 expression).

[0007] Figures 2A-2C The functional affinity of TCR 11NA4 (see Table 1) is compared with that of TCR 220_21 (V domain amino acid sequences shown in SEQ ID NO.: 61 (Vα) and 62 (Vβ)) and TCR "BNT" (Vα domain amino acid sequence shown in SEQ ID NO.: 60 (including signal peptide); Vβ domain amino acid sequence shown in SEQ ID NO.: 59 (including signal peptide)). ( Figure 2A ) Percentage of primary CD8+ T cells transduced with TCR expressing CD137 in the presence of KRAS G12V peptide at the indicated concentrations; ( Figure 2B ) log EC50 of TCR for KRAS G12V peptide; ( Figure 2C ) Percentage of TCR-transduced primary CD8+ T cells expressing IFN-γ in the presence of the indicated concentrations of KRAS G12V peptide.

[0008] Figures 2D-2F The functional avidity of TCR 11N4A (assessed as CD137 expression determined by host T cells) is shown. Figure 2D showed that T cells transduced with TCR 11N4A recognized KRAS G12V 9-mer and 10-mer peptides; Figure 2E The log EC50 of TCR for KRAS G12V 9-mer (left) and 10-mer (right) peptides is shown; values ​​refer to Figure 2D and shows the strong functional affinity of TCR 11N4A (low picomolar EC50); and Figure 2FThis shows that TCR 11N4A does not recognize the KRASG12G wild-type peptide. Figure 2E The y-axis values ​​on the chart (from bottom to top) are: -11, -10, -9, -8, -7, -6, -5. Figure 2F In the chart, the y-axis values ​​(from bottom to top) are: 0, 20, 40, 60, 80, 100; the x-axis text (from left to right) is: No peptide; G12V 7-18 10-mer; G12V 8-18 9-mer; G12G 7-18 WT;G12G 8-18 WT.

[0009] Figure 2G It was shown that transduced T cells expressing TCR 11N4A were specifically activated in response to mutant KRAS G12V peptide, but not in response to wild-type KRAS G12G 9-mer or 10-mer peptide. A population of sorted and purified donor T cells expressing TCR 11N4A was exposed to mutant KRAS G12V 9-mer, wild-type KRASG12G 9-mer or 10-mer, or no peptide control at 1 μg / mL for 16 hours, and T cell activation was assessed by CD137 (4-1BB) expression.

[0010] Figures 3A-3G Shown is the recognition of tumor cell lines expressing HLA-A11+KRAS G12V by TCR-transduced T cells (assessed by the percentage of TCR-transduced T cells expressing CD137). "UT" = untransduced, negative control. Figure 3C Activation of untransduced T cells or T cells engineered with 11N4A-TCR and CD8αβ co-receptor in different tumor cell lines by endogenous KRAS G12V presentation is shown.

[0011] exist Figures 3D-3F In the figure, 11N4A-TCR T cells are shown as “FH-KRAS-TCR”. Figure 3D Figure 1 shows activation of 11N4A-TCR cells by endogenously processed and presented KRAS G12V antigen in a panel of different tumor cell lines. (Left) Description of the tumor cell lines used in this study. (Right) * The indicated tumor cell lines expressing 11N4A and KRAS G12V were cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) at an effector to target ratio of 1:1 for 20 h. T cell activation was measured by flow cytometry with CD137 surface staining. Figure 3E11N4A-TCR cells were shown to secrete effector cytokines in response to endogenously processed and presented KRAS G12V antigen in a panel of different tumor cell lines. * The indicated tumor cell lines expressing 11N4A and KRAS G12V were cultured with untransduced or 11N4A-TCR T cells from two healthy donors (D1 and D2) for 20 h. Figure 3D Supernatants from co-culture activation assays were analyzed by ELISA for IFNγ, TNFα, and IL-2 cytokine secretion as indicated. IU = International Units calculated based on a standard curve of IFNγ recombinant protein. Figure 3F 11N4A-TCR T cells were shown to proliferate in response to endogenously processed and presented KRAS G12V antigen in a panel of different tumor cell lines. * The indicated tumor cell lines expressing 11N4A 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 flow cytometry lymphocyte counts. T cell counts are plotted as lymphocyte counts / μL. Figure 3G Figure 2 shows a panel of different tumor cell lines tested that displayed 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 for cell number. G12V Specific antibody (MA5-42375, Invitrogen), with GAPDH-specific antibody used as loading control (AB9483, Abcam). (Right) Densitometric analysis of the left western blot data, quantifying the ratio of KRAS G12V expression to GAPDH expression for all tested tumor cell lines.

[0012] Figures 4A-4G Specific killing of HLA-A11+KRAS G12V-expressing tumor cell lines by CD8+ T cells expressing KRAS G12V-specific TCR in the Incuyte killing assay. In this assay, red object areas indicate the presence of tumor cells. Figure 4A ) Schematic diagram of mKRAS tumor cell growth in the absence of mKRAS-specific T cells. ( Figure 4B ) Growth curves of mKRAS+ / HLA-A11+ tumor cells in a killing assay. Test conditions included tumor cells alone, tumor cells + T cells transduced to express TCR 11N4A, and tumor cells transduced to express the comparator TCR 220_21. The red area on the y-axis shows tumor cell growth. Additional tumor cells were added at 72 hours. Figure 4C ) Data from another killing assay. T cells and SW480 tumor cell line were present at the indicated effector:target ratios. ( Figure 4D ) 11N4A-transduced primary CD4+ and CD8+ T cells were cytotoxic to SW527, SW620, and CFPAC-1 tumor cell lines across multiple tumor cell lines. In the presence of 11N4A-transduced (lower curve in each graph) or non-transduced (upper curve in each graph) primary T cells, the indicated HLA-A * Growth kinetics of 11:01+, KRAS G12V-expressing tumor cell lines. 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 a 10:1 effector to target ratio for approximately 145 hours, as indicated. Tumor confluence is reported as a measure of tumor cell growth / viability throughout the study. Additional tumor cells were added at approximately 50 and 90 hours. Figure 4E )-( Figure 4G ): 11N4A-TCR cells are cytotoxic to a panel of different tumor cell lines. In a live tumor visualization assay, various indicated HLA-A * Growth kinetics of 11:01+, KRAS G12V-expressing tumor cell lines. 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. As shown, throughout the study, tumor cell confluence, as measured by the total red object area (marked as tumor cell confluence), was reported as a measure of tumor cell growth / viability. In (E)-(G), 11N4A-TCR T cells are shown as "FH-KRAS-TCR".

[0013] Figures 5A-5D Involves mutagenesis scanning experiments using KRAS G12 9-mer and 10-mer peptides to characterize the peptide binding motif of TCR 11N4A. Figure 5A) The percentage of T cells transduced by TCR expressing Nur77-GFP in the presence of G12V peptide (shown as "G12V WT") or variants of the G12V polypeptide in which the amino acid at the indicated position was replaced by alanine, glycine, or threonine, as indicated. Top: Results of mutation scanning of the KRAS G12 9-mer peptide. Bottom: Results of mutation scanning of the KRAS G12 10-mer peptide. ( Figure 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 ) Schematic diagram of the workflow used to identify sequences from the human proteome containing sequences similar to the TCR 11N4A binding motif, and the results of the workflow. ( Figure 5D )use Figure 5C Results of the workflow shown for searching the human proteome. Peptides from the human proteome were scored for their predicted binding to HLA-A11.

[0014] Figures 6A-6G It was shown that TCR 11N4A has a low risk of autoreactivity in humans. ( Figure 6A 、 Figure 6B ) Reactivity of 11N4A-transduced T cells to a panel of potentially cross-reactive peptides (see Figure 5B ). ( Figure 6C ) peptide dose response curves and ( Figure 6D ) Calculated negative log EC50 of 11N4A-transduced T cells to RAB7B peptide and cognate KRAS G12V peptide. ( Figure 6E ) The percentage of 11N4A-transduced CD8+ T cells expressing CD137 in response to overnight cultures with a comprehensive panel of positional scanning peptides containing substitutions of every possible amino acid at every position of the homologous KRAS G12V peptide (172 peptides). Peptides that elicited greater than 15% responses were considered positive in this assay. ( Figure 6F )(Left) Search from ScanProsite from ( Figure 6E (Right) CD137 expression (determined by flow cytometry) of primary CD8+ T cells purified by sorting, transduced to express TCR 11N4A or TCR 11N4A + CD8αβ, and cultured overnight with 100 ng / ml of potentially cross-reactive peptides. Figure 6G) TCR11N4A does not cause cross-reactive peptide responses in vitro. T cell activation assay performed using T cells stimulated with 11N4A-TCR peptides generated from PBMCs of two healthy donors. TCR 11N4A-T cells were incubated with 1 μg / ml of each designated peptide for 18 hours, followed by flow cytometric analysis of CD137 expression as a measure of T cell activation. (Left) Human autologous peptides identified from alanine scanning motifs and (right) human autologous peptides identified from XScan scanning motifs (see Table 2).

[0015] Figure 7A and 7B Screening involving evaluation of potential alloreactivity of TCR 11N4A. Figure 7A ) B lymphoblastoid cell lines (B-LCL) expressing different HLA alleles were incubated with 11N4A-transduced CD8+ T cells, and T cell responsiveness was assessed as determined by expression of IFN-γ or CD137. ( Figure 7B ) Results of alloreactivity screening: Percentage of CD137+11N4A-transduced T cells with (top) or without (bottom) CD8αβ) against B-LCL expressing common HLA alleles.

[0016] Figure 8 The killing activity of CD8+ and CD4+ T cells engineered to express TCR 11N4A and CD8αβ co-receptors against mKRAS:HLA-A11+ tumor cells is shown.

[0017] Figures 9A-9J Nucleotides associated with TCR 11N4A are shown ( Figures 9A-9G ) and amino acids ( Figures 9H-9J ) sequences and expression constructs encoding or comprising them.

[0018] Figures 10A-10H Nucleotides associated with TCR 11N6 are shown ( Figures 10A-10E ) and amino acids ( Figures 10F-10H ) sequences and expression constructs encoding or containing them.

[0019] It should be understood that it is not Figures 9A-10H All sequences shown in the Figure legends contain or are annotated with each of the sequence features indicated in the figure legends. CDR3 sequences are shown according to the IMGT junction definition.

[0020] Figures 11A-11D 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 ). T cells ("E") and target cells ("T") are at the specified E:T ratios.

[0021] Figures 12A-12C Shown in SW527( Figure 12A )、CFPAC( Figure 12B ) and SW620( Figure 12C ) Robust in vivo antitumor activity of primary CD4+ and CD8+ T cells engineered to express the 11N4A TCR and CD8αβ co-receptor in a tumor challenge model.

[0022] Figures 13A-13E It was demonstrated that the in vitro and in vivo anti-tumor efficacy was enhanced by combined treatment of 11N4A TCR and CD8αβ co-receptor engineered CD4+ T cells and 11N4A TCR and CD8αβ co-receptor engineered CD8+ T cells compared to single treatment of 11N4A TCR and CD8αβ co-receptor engineered CD4+ T cells or 11N4A TCR and CD8αβ co-receptor engineered CD8+ T cells. Figure 13A ) The growth kinetics of the CFPAC-1 tumor cell line expressing HLA-A11+KRAS G12V were measured in the presence of 11N4A TCR-CD8αβ coreceptor engineered CD4+ T cells, 11N4A TCR-CD8αβ coreceptor engineered CD8+ T cells, or 11N4ATCR-CD8αβ coreceptor engineered CD4+ and CD8+ T cells; ( Figure 13B Tumor kinetics of CFPAC-Luc tumor cells inoculated in NSG immunocompromised mice were measured after intraperitoneal treatment with untransduced CD4+ and CD8+ T cells, 11N4A TCR-CD8αβ coreceptor-engineered CD4+ T cells, 11N4ATCR-CD8αβ coreceptor-engineered CD8+ T cells, or 11N4ATCR-CD8αβ coreceptor-engineered CD4+ and CD8+ T cells. Figure 13C )-( Figure 13E ): The combination of CD8αβ co-receptor (also written as "CD8α / β" co-receptor) and class I TCR improves the anti-tumor response of TCR-engineered T cells. + T cells, CD8 only + T cells or TCR 11N4A ( Figure 13C ) or TCR 11N4 A+CD8αβ co-receptor ( Figure 13D ) transduced CD4 + / CD8 + In the presence of T cells (1:1 ratio), HLA-A* 11:01 + , growth kinetics of tumor cell line (OVCAR-5) expressing KRASG12V. Negative control tumor cell line (PANC1) not expressing KRASG12V was observed in ( 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, as measured by NucLight Red total red object area, was reported as a measure of tumor cell growth / viability throughout the study, as indicated. Additional tumor cells were added at 72 and 108 hours.

[0023] Figure 14 It was shown that T cells transduced with TCR 11N4A and CD8αβ co-receptor did not display cytokine-independent growth in vitro.

[0024] Figure 15 It is shown that the T cell products simulated by 11N4A-TCR do not respond to the RAB7B peptide. The peptide doses of KRAS G12V index peptide (positive control) and RAB7B in the range of 10-0.00001 mg / mL were tested to evaluate the reactivity of the T cell products simulated by 11N4A-TCR produced by two donors. 9-mer and 10-mer KRAS G12V and RAB7B peptides were exogenously added to 11N4A-TCR T cells at titrated doses for 16 hours, and then the CD137 expression of T cells was analyzed by flow cytometry.

[0025] Figure 16 The 11N4A-TCR mimics a T cell product that does not respond 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 protein. + ) and PANC1(KRAS G12V - ) were used as positive and negative controls, respectively. + or CD8 + 11N4A-TCR or untransduced (UTD) T cell products were co-cultured with each cell line for 16 hours, and then T cells were analyzed by flow cytometry for CD137 expression. In this figure, 11N4A-TCR T cells are shown as "FH-KRAS-TCR."

[0026] Figure 17It is shown that in the absence of cytokines, the product of 11N4A-TCR simulation does not show cell growth. 11N4A-TCR T cell products are enriched for KRAS-G12V A11 tetramer-positive T cells and expanded for 10 days in two donors using X VIVO 15+5% serum replacement medium+100U / mL IL-2 in anti-CD3 and anti-CD28 beads. Untransduced primary T cells (UTD) from the same donor were similarly expanded side by side for 10 days. On day 10, the cells were washed and resuspended in X VIVO 15+5% serum medium lacking cytokines, and cell growth kinetics were measured over 35 days. Figure 17 In the figure, 11N4A-TCR T cells are shown as “FH-KRAS-TCR”. DETAILED DESCRIPTION

[0027] The present disclosure generally relates to binding proteins specific for Ras neoantigens, modified host (e.g., immune) cells expressing the binding proteins, polynucleotides encoding the binding proteins, and related uses. Mutated Ras proteins (e.g., KRAS, NRAS, HRAS) can produce neoantigens, including a G→V mutation at position 12 of the full-length KRAS protein (SEQ ID NO.: 1; UniProtKB 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).

[0028] In the present disclosure, binding proteins capable of binding to Ras neoantigens are provided. In certain aspects, binding proteins comprising a TCR Vα domain and a TCR Vβ domain (and a host cell, such as an immune cell, comprising a heterologous polynucleotide encoding a Ras-specific binding protein of the present disclosure) are provided, wherein the binding protein is 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 shown in any one of SEQ ID NO: 2 or 3. In certain embodiments, the HLA comprises HLA-A * 11, such as HLA-A * 11:01.

[0029] The disclosed binding proteins are highly sensitive to antigens, and in certain embodiments the binding proteins are capable of inducing activation of host T cells at low concentrations of peptide antigens. In certain embodiments, in a population or sample of (e.g., CD8+ or CD4+) T cells expressing a binding protein, when there is [LogEC50 less than -9M (e.g., between -9M and -10M)] peptide, T cells have half-maximal expression of the activation marker Nur77. In certain embodiments, in a population or sample of (e.g., CD8+ or CD4+) T cells expressing a binding protein, when there is [LogEC50 less than -10M (e.g., between -10M and -11M)] peptide, T cells have half-maximal expression of CD137. In certain embodiments, in a population or sample of (e.g., CD8+ or CD4+) T cells expressing a binding protein, when there is [LogEC50 less than -10M (e.g., between -10M and -11M)] peptide, T cells have half-maximal expression of IFN-γ.

[0030] Host (e.g., T) cells expressing a binding protein according to the present disclosure are activated (e.g., as determined by expression of CD137) in the presence of cancer cell lines expressing mutant KRAS, 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.

[0031] 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 are capable of specifically killing cells expressing mutant KRAS (e.g., SW480 cells, such as at an 8:1 effector:target ratio, a 4:1 effector:target ratio, or a 2:1 effector:target ratio) in vitro for more than 144 hours, including when additional tumor cells (i.e., additional cells expressing mutant KRAS) are added at 72 hours in a rechallenge setting.

[0032] In certain embodiments, the binding proteins of the present disclosure are non-alloreactive, substantially non-alloreactive, and / or have a lower risk of alloreactivity to (i) amino acid sequences from the human proteome and / or (ii) human HLA alleles.

[0033] In any of the embodiments disclosed herein, the binding protein may be human, humanized or chimeric. Also provided are polynucleotides encoding the binding protein, vectors comprising the polynucleotides, and host cells comprising the polynucleotides and / or vectors and / or expressing the binding protein. Currently disclosed binding proteins and host cells (e.g., T cells, NK cells, NK-T cells) can be used to treat diseases or conditions associated with KRAS neoantigens, for example, cancer. Currently disclosed binding proteins can also bind to the G12V antigen produced in human NRAS or human HRAS, which contains the same sequence as KRAS in the region near the G12 residue. Therefore, the disclosed compositions can be used to treat diseases or conditions associated with KRAS neoantigens comprising G12V mutations, NRAS neoantigens comprising G12V mutations, or HRAS neoantigens comprising G12V mutations, or any combination thereof.

[0034] Also provided are methods and uses of the presently disclosed binding proteins, polynucleotides, vectors, host cells, and related compositions for treating a disease or disorder associated with a KRAS, NRAS, and / or HRAS mutation as provided herein.

[0035] Also provided are methods comprising introducing a polynucleotide encoding a presently disclosed binding protein (or introducing a vector comprising the polynucleotide) into a host cell or multiple host cells or a population or sample of host cells. In some embodiments, the polynucleotide or vector further encodes: a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain; a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain, or both. In certain embodiments, the one or more host cells comprise T cells, such as CD4+ T cells or CD8+ T cells. In certain embodiments, the one or more host cells comprise primary T cells. In certain embodiments, the one or more host cells comprise peripheral blood mononuclear cells (PBMCs). In some embodiments, the method further comprises culturing the one or more host cells. In some embodiments, the one or more host cells are from a subject suffering from a disease or condition associated with a KRAS G12V, NRAS G12V, or HRAS G12V mutation. In some embodiments, the disease or condition comprises cancer. In some embodiments, the subject is positive for expression of HLA-A11, such as HLA-A*11:01. In certain embodiments, the one or more host cells 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 prepared by the methods. In some embodiments, the host cell population comprises CD8+ T cells, CD4+ T cells, or both. In some embodiments, the methods further comprise selecting CD8+ T cells and CD4+ T cells and combining them to provide a composition comprising CD8+ T cells and CD4+ T cells at a ratio of about 1:1.

[0036] Before describing the present disclosure in more detail, it may be helpful to understand the present disclosure by providing definitions of certain terms used herein. Additional definitions are set forth throughout the present disclosure.

[0037] In this specification, unless otherwise stated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer in the scope, and its fraction (such as one tenth and one hundredth of an integer) when appropriate. Moreover, unless otherwise stated, any number range related to any physical characteristics such as polymer subunits, size or thickness set forth herein should be understood to include any integer in the scope stated. As used herein, unless otherwise stated, the term "about" means ± 20% of the indicated range, value or structure. It should be understood that, as used herein, the term "one / a kind (a)" and "one / a kind (an)" refer to "one or more" in the listed components. The use of alternatives (for example, "or") should be understood to mean one, two or any combination thereof in the alternatives. As used herein, the terms "include," "have" and "comprise" are used synonymously, and the terms and their variants are intended to be interpreted as non-restrictive.

[0038] In addition, it should be understood that this application discloses individual compounds or groups of compounds derived from various combinations of structures and substituents described herein to the same extent as if each compound or group of compounds were set forth individually. Therefore, the selection of a particular structure or a particular substituent is within the scope of this disclosure.

[0039] The term "consisting essentially of" is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those materials or steps 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 a protein (which can have one or more domains, regions or modules) "consists essentially of" a particular amino acid sequence when the amino acid sequence of the domain, region, module or protein includes extensions, deletions, mutations or a combination thereof (e.g., at the amino or carboxyl termini or between domains) that together account for up to 20% (e.g., up to 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., do not reduce the 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, region, module or protein (e.g., the target binding affinity or avidity of the binding protein).

[0040] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Protein applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as non-naturally occurring amino acid polymers. In some embodiments, "peptide" (e.g., peptide antigen) refers to a polymer of about 8-10 amino acid residues in length.

[0041] As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from a hematopoietic stem cell or fetal tissue and is capable of further differentiating into a mature cell type (e.g., an immune system cell). Exemplary hematopoietic progenitor cells include those with CD24 Lo Lin – CD117 + phenotype of cells or cells found in the thymus (called progenitor thymocytes).

[0042] As used herein, "immune system cell" means any cell of the immune system that is derived from hematopoietic stem cells in the bone marrow, which give rise to two major lineages, myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, gamma delta T cells, regulatory T cells, natural killer cells, natural killer T cells, and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs." They are specialized cells that can activate T cells when peptide-complexed major histocompatibility complex (MHC) receptors on the surface of the APC interact with the TCR on the surface of the T cell.

[0043] "T cells" or "T lymphocytes" are cells of the immune system that mature in the thymus and produce T cell receptors (TCRs). T cells can be naive T cells ("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, 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, with decreased expression of CD62L, CCR7, CD28 and CD45RA). Effector T cells (TE) refer to antigen-experienced CD8+ cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7 and CD28 compared to TCM, and are positive for granzymes and perforins (e.g., upon stimulation). Helper T cells (T H ) is CD4 + CD4 cells, which influence the activity of other immune cells by releasing cytokines. + T cells can activate and suppress adaptive immune responses, and which of these two functions is induced will depend on, for example, 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 using known techniques, such as by affinity binding with antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4 T cells. + CD25 + (Foxp3 + ) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8 + CD28 - and Qa-1-restricted T cells.

[0044] A "T cell receptor" (TCR) refers to a member of the immunoglobulin superfamily having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail that is capable of specifically binding an antigenic peptide bound to an MHC receptor; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, p. 433, 1997). TCRs can be present on the cell surface or in a soluble form and typically comprise heterodimers having α and β chains (referred to as TCRα and TCRβ, respectively) or γ and δ chains (referred to as TCRγ and TCRδ, respectively). In certain embodiments, polynucleotides encoding the binding proteins (e.g., TCRs) of the present disclosure may be codon-optimized to enhance expression in specific 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 that can express the binding proteins and TCRs of the present disclosure include CD4 T cells, ... + T cells, CD8 + T cells and their related subsets (e.g., naive, central memory, stem cell memory, effector memory).

[0045] As with immunoglobulins (e.g., antibodies), the extracellular portion of a TCR chain (e.g., α chain, β chain) can contain two immunoglobulin domains at the N-terminus, a variable domain (e.g., α chain variable domain or Vα, β chain variable domain or Vβ; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.)), and a constant domain adjacent to the cell membrane (e.g., α chain constant domain or Cα, typically 5 amino acids 117 to 259 based on Kabat, β chain constant domain or Cβ, typically amino acids 117 to 295 based on Kabat). In addition, like immunoglobulins, the variable domain contains 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., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). TCRs used in the present disclosure can be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0046] The term "variable region" or "variable domain" refers to a domain of an immunoglobulin superfamily binding protein (e.g., TCR α chain or β chain (or γ chain and δ chain of γδTCR) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., TCR) to an antigen. The variable domains of the α chain and β chain of a natural TCR (Vα and Vβ, respectively) generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three CDRs. The Vα domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ); the Vβ domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). A single Vα or Vβ domain may be sufficient to confer antigen binding specificity. In addition, TCRs that bind to a specific antigen can be isolated from a library of TCRs that bind to the antigen to screen for complementary Vα or Vβ domains, respectively.

[0047] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to amino acid sequences within the variable region of an immunoglobulin (e.g., TCR). CDRs confer antigen specificity and binding affinity and are separated from each other in the primary amino acid sequence by framework regions. In general, there are three CDRs in each TCR α chain variable region (αCDR1, αCDR2, αCDR3 (also identified as CDR1α, CDR2α, and CDR3α, respectively)) and three CDRs in each TCR β chain variable region (βCDR1, βCDR2, βCDR3 (also identified as CDR1β, CDR2β, and CDR3β, respectively)). In TCRs, CDR3 is considered to be the primary CDR responsible for recognizing processed antigens. In general, CDR1 and CDR2 primarily or exclusively interact with MHC.

[0048] CDR1 and CDR2 are encoded within the variable gene segments of the TCR variable region encoding sequence, while CDR3 is encoded by a region spanning the Vα variable and joining segments, or by a region spanning the Vβ variable, diversity, and joining segments. Thus, if the identity of the variable gene segments of Vα or Vβ is known, the sequences of their corresponding CDR1 and CDR2 can be inferred; for example, according to the numbering scheme described herein. Compared to CDR1 and CDR2, CDR3, and particularly CDR3β, typically has significantly greater diversity due to the addition and loss of nucleotides during the recombination process.

[0049] TCR variable domain sequences can be aligned with numbering schemes (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), allowing for annotation of equivalent residue positions and comparison of different molecules using, for example, the ANARCI software tool (2016, Bioinformatics 15: 298-300). The numbering scheme provides standardized definitions 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., Development Compet. Immunol. 27: 55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, CDRs (e.g., CDR3) are identified or defined according to the IMGT engagement definition. In some embodiments, CDRs (e.g., CDR3 or all six CDRs of a binding protein) are 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 present 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 present 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 present 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 present disclosure (or all six CDRs of a binding protein) are identified or defined according to the enhanced Chothia numbering scheme or method. In some embodiments, the CDRs of the present disclosure (or all six CDRs of a binding protein) are identified or defined according to the Aho numbering scheme or method.

[0050] The source of TCR as used in the present disclosure can be from any of various animal species, such as humans, mice, rats, rabbits or other mammals. The TCR constant domain sequence can be from, for example, humans, mice, marsupials (e.g., opossums, bandicoots, wallabies), sharks or non-human primates. In certain preferred embodiments, the TCR constant domain sequence is human or comprises an engineered variant of a human sequence. The TCR constant domain can be engineered to, for example, improve pairing, expression, stability or any combination of these. 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 of TCR Cα and Cβ include mutating natural amino acids to cysteine, thereby forming a disulfide bond between the introduced cysteine ​​of one TCR constant domain and the natural cysteine ​​of another TCR constant domain. Such mutations may include, for example, T48C in Cα, T57C or S57C in Cβ, or both. An embodiment is also provided in which a homologous TCR constant domain comprises a mutation, 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 natural amino acids with one or more amino acids having smaller side chains), and another (e.g., another of Cα and Cβ) comprises a compensatory so-called "protuberance" (e.g., obtained by replacing one or more natural amino acids with one or more amino acids having larger side chains), similar to a "knob-and-hole" configuration for promoting preferential pairing of antibody heavy chains. An embodiment in which TCR constant domain amino acids are mutated to introduce or modify charge properties to facilitate the pairing of mutated constant domains is also provided. Examples of mutations that can occur in Cα and Cβ to promote specific pairing by a knob-in-hole mechanism or by a charge pairing mechanism 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. Pat. No. 9,062,127. TCR constant domain mutations, mutated TCR constant domains, and methods for identifying sites for mutation are described in these documents and are incorporated herein by reference.

[0051] Mutations that increase stability may include a mutation in the Ca transmembrane domain from the sequence LSVIGF to the sequence LLVIVL ("LVL" mutation; see Haga-Friedman et al., J. Immunol. 188:5538-5546 (2012), which TCR mutations and mutant TCR constant domain sequences are incorporated herein by reference).

[0052] As used herein, the term "CD8 co-receptor" or "CD8" refers to the cell surface glycoprotein CD8, whether α-α homodimer or α-β heterodimer. The CD8 co-receptor contributes to the function of cytotoxic T cells (CD8+) and exerts its effect through signal transduction of its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, "Immunol. Today" 21:630-636, 2000; Cole and Gao, "Cell. Mol. Immunol." 1:81-88, 2004). There are five (5) human CD8 β chain isotypes (see UniProtKB identifier P10966) and a single human CD8 α chain isotype (see UniProtKB identifier P01732).

[0053] “CD4” is an immunoglobulin co-receptor glycoprotein that facilitates the communication between the TCR of CD4+ cells and antigen-presenting cells (see Campbell and Reece, Biology )》909 (Benjamin Cummings, 6th edition, 2002). CD4 is present on the surface of immune cells such as T helper cells, monocytes, macrophages and dendritic cells, and includes four immunoglobulin domains (D1 to D4) expressed on the cell surface. During antigen presentation, CD4 is recruited along with the TCR complex to bind to different regions of the MHCII molecule (CD4 binds to MHCII β2, while the TCR complex binds to MHCII α1 / β1). Without wishing to be bound by theory, it is believed that close proximity to the TCR complex allows the CD4-associated kinase molecule to phosphorylate the immunoreceptor tyrosine-based activation motif (ITAM) present on the cytoplasmic domain of CD3. This activity is thought to amplify the signal generated by the activated TCR in order to generate or recruit various types of immune system cells, including T helper cells and immune responses.

[0054] In certain embodiments, TCR is present on the surface of T cells (or T lymphocytes) and associates with CD3 complexes." CD3 " is a six-chain multiprotein complex (see, Abbas and Lichtman, 2003; Janeway et al., pages 172 and 178, 1999), which is associated with antigen signaling in T cells. In mammals, the complex comprises a homodimer of CD3γ chain, CD3δ chain, two CD3ε chains and CD3ζ chain. CD3γ, CD3β and CD3ε chain are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane region of CD3γ, CD3β and CD3ε chains is negatively charged, which is believed to allow these chains to associate with the positively charged region of T cell receptor chains. The intracellular tails of CD3γ, CD3β and CD3ε chains each contain a single conserved motif, referred to as an activation motif or ITAM based on immunoreceptor tyrosine, and each CD3ζ chain has three conserved motifs. Without wishing to be bound by theory, it is believed that ITAMs are important for the signaling ability of the TCR complex.The CD3 used in the present disclosure can be from various animal species, including humans, mice, rats or other mammals.

[0055] As used herein, "TCR complex" refers to a complex formed by the association of CD3 and TCR. For example, the TCR complex can be composed of the following: 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 can be composed of the following: a CD3γ chain, a CD3β chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRβ chain.

[0056] As used herein, a "component of a TCR complex" 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 sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0057] A "chimeric antigen receptor" (CAR) refers to a fusion protein that has been engineered to contain two or more naturally occurring amino acid sequences, domains, or motifs, linked together in a manner that does not occur naturally or naturally in a host cell, and that can function as a receptor when the fusion protein is present on the surface of a cell. A CAR can include an extracellular portion comprising an antigen binding domain (e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR specific for a cancer antigen or a TCR binding domain obtained therefrom, an antibody or a scFv obtained therefrom, or a killer immune receptor derived from a NK cell or an antigen binding domain obtained therefrom) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing a costimulatory domain) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al., Cancer Immunol. Immunother. 63(11):1163 (2014), and Walseng et al., Scientific Reports). (Reports)》7:10713(2017), wherein CAR constructs and methods for preparing the same are incorporated herein by reference). The CAR of the present disclosure that specifically binds to the Ras antigen (e.g., in the context of a peptide:HLA complex) comprises a TCR Vα domain and a Vβ domain.

[0058] Any polypeptide of the present disclosure, as encoded by a polynucleotide sequence, may comprise a "signal peptide" (also referred to as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to an appropriate location within or outside the cell (e.g., to be inserted or positioned in the cell membrane, or to be secreted by the cell, or to be contained within the cell). In some contexts, the length of the signal peptide is about 15 to about 22 amino acids. During or once the positioning (e.g., membrane insertion) or secretion is completed, the signal peptide can be removed from the polypeptide. In some embodiments, the signal peptide is completely removed from the polypeptide. In some embodiments, a signal peptide of less than all but generally no more than one, two, three, four, five, or six amino acids is retained in the polypeptide, and the remaining signal peptides are removed. Polypeptides with signal peptides are referred to herein as "preproteins," and polypeptides from which their signal peptides are removed are referred to herein as "mature" proteins or polypeptides. In any of the embodiments disclosed herein, the binding protein or fusion protein comprises or is a mature protein, or or comprises a preprotein.

[0059] "Linker" refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs, and can provide a spacer function compatible with the interaction of two sub-binding domains so that the resulting polypeptide maintains specific binding affinity to the target molecule (e.g., scTCR) or maintains signal transduction activity (e.g., TCR complex). In certain embodiments, the linker comprises about two to about 35 amino acids, for example, or about four to about 20 amino acids, or about eight to about 15 amino acids or about 15 to about 25 amino acids. Exemplary linkers include glycine-serine linkers.

[0060] As used herein, "antigen" or "Ag" refers to an immunogenic molecule that causes an immune response. This immune response can involve the production of antibodies, the activation of specific immune competent cells, or both. Antigens (immunogenic molecules) can be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, etc. It is apparent that antigens can be synthesized, recombinantly produced, or derived from biological samples. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express antigens, or by cells that express immunogenic mutations or polymorphisms endogenously (e.g., without modification or genetic engineering of human intervention).

[0061] As used herein, "neoantigen" refers to a host cell product containing a structural change, alteration or mutation that produces a new antigen or antigenic epitope that was not previously observed in the subject's genome (i.e., in a sample of healthy tissue from the subject) or that is "seen" or recognized by the host immune system, the new antigen: (a) processed by the cell's antigen processing and transport machinery and presented on the surface of the cell in association with MHC (e.g., HLA) molecules; and (b) triggering an immune response (e.g., a cellular (T cell) response). Neoantigens can be derived from, for example, polynucleotides encoding changes (substitutions, additions, deletions) that result in altered or mutated products, or from inserting exogenous nucleic acid molecules or proteins into cells, or from exposure to environmental factors (e.g., chemicals, radioactivity) that result in genetic changes. Neoantigens can be produced separately from tumor antigens, or can be produced by or associated with tumor antigens. "Tumor neoantigens" (or "tumor-specific neoantigens") refer to proteins that contain neoantigenic determinants associated with, produced by, or produced within tumor cells or multiple cells within a tumor. Tumor neoantigenic determinants are present, 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 from viral open reading frames associated with virus-associated tumors (e.g., cervical cancer, some head and neck cancers). When referring to Ras antigens comprising mutations as disclosed herein, the terms "antigen" and "neoantigen" are used interchangeably herein.

[0062] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, a T cell receptor (TCR), a chimeric antigen receptor, or other binding molecule, domain, or protein. Epitope determinants typically contain chemically active surface groupings of molecules such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics and specific charge characteristics.

[0063] As used herein, the term "KRAS (or NRAS or HRAS) antigen (or neoantigen)" or "KRAS (or NRAS or HRAS) peptide antigen (or neoantigen)" or "KRAS (NRAS or HRAS) peptide" refers to a naturally or synthetically produced peptide portion of a KRAS or NRAS or HRAS protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids, up to about 20 amino acids, and comprising at least one amino acid change caused by a G12 (e.g., G12V) mutation (wherein position 12 refers to the full-length KRAS protein sequence set forth in SEQ ID NO: 1; and also refers to the full-length NRAS and HRAS protein sequences set forth in SEQ ID NOs.: 78 and 79, respectively), said peptide can form a complex with an MHC (e.g., HLA) molecule, and the binding proteins of the present disclosure that are specific for KRAS or NRAS or HRAS peptide:MHC (e.g., HLA) complexes can specifically bind to such complexes. Exemplary KRAS (or NRAS or HRAS) antigens comprise, consist of, or consist essentially of a peptide having the amino acid sequence of SEQ ID NO.: 2 or 3.

[0064] The "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 with a transmembrane α chain (with three α domains) and a non-covalently associated β2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain contains two domains. MHC class I molecules deliver peptides derived from the cytosol to the cell surface where the peptide:MHC complex is bound by CD8 + T cell recognition. MHC class II molecules deliver peptides derived from the vesicle system to the cell surface where they are recognized by CD4 + T cell recognition. Human MHC is called human leukocyte antigen (HLA). HLA corresponding to "class I" MHC presents peptides from cells and includes, for example, HLA-A, HLA-B, and HLA-C. Alleles include, for example, HLA A * 11, such as HLA-A * 11: 01. HLA corresponding to "class II" MHC presents peptides from outside the cell and includes, for example, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0065] The principles of antigen processing by antigen-presenting cells (APCs) (such as dendritic cells, macrophages, lymphocytes, or other cell types) and antigen presentation by APCs to T cells, including immunocompatibility (e.g., sharing at least one allelic form of an MHC gene involved in antigen presentation) between APCs and T cells, are well established (see, e.g., Murphy, Janeway's Immunobiology (8th ed.), Garland Science, NY, 2011; Chapters 6, 9, and 16). For example, processed antigenic peptides originating from the cytosol (e.g., tumor antigens, intracellular pathogens) will typically be about 7 to about 11 amino acids in length and will associate with class I MHC (HLA) molecules, whereas peptides processed in vesicular systems (e.g., bacteria, viruses) will typically vary in length from about 10 to about 25 amino acids and associate with class II MHC (HLA) molecules.

[0066] As used herein, the term "KRAS-specific binding protein" refers to a protein or polypeptide, such as a TCR, scTv, scTCR, or CAR, that binds to a KRAS peptide antigen or a NRAS peptide antigen or a HRAS peptide antigen (or to a KRAS or NRAS or HRAS peptide antigen:HLA complex, e.g., on a cell surface), and does not bind to peptides that do not contain a KRAS or NRAS or HRAS peptide antigen, and does not bind to an HLA complex containing such peptides.

[0067] The binding proteins of the present disclosure, such as TCR, scTv, scTCR and CAR, contain binding domains that are specific for the target. As used herein, "binding domain" (also referred to as "binding region" or "binding portion") refers to a molecule or part thereof (e.g., peptide, oligopeptide, polypeptide, protein) that has the ability to associate, combine or combine with a target (e.g., KRAS or NRAS or HRAS peptide or KRAS or NRAS or HRAS peptide: MHC complex) specific and non-covalently associate, combine or combine. Binding domain includes any naturally occurring, synthetic, semi-synthetic or recombinantly produced binding partner of a biomolecule, a molecular complex (i.e., a complex comprising two or more biomolecules) or other target of interest. Exemplary binding domains include immunoglobulin variable regions or single-chain constructs (e.g., single-chain TCR (scTCR) or scTv) comprising the immunoglobulin variable region.

[0068] In certain embodiments, the Ras-specific binding protein is expressed in an amount less than about 10 -8 M, less than about 10 -9 M, less than about 10-10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M's K d , or binds to a KRAS (or NRAS or HRAS) peptide (or KRAS (or NRAS or HRPS):HLA complex) with an affinity that is about the same as, at least about the same as, or greater than, or about the affinity exhibited by an exemplary Ras-specific binding protein 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 a binding portion thereof.

[0069] As used herein, "specifically binds to" or "has specificity for" refers to the association or binding of a binding protein (e.g., a TCR receptor) or binding domain (or a fusion protein thereof) to a target molecule with an affinity or K a (i.e., the equilibrium association constant for a specific binding interaction, expressed in 1 / M) equal to or greater than 10 5 M -1 (which is equal to the association rate of this association reaction [k on ] and the dissociation rate [k off ] ratio) without significantly associating or combining with any other molecule or component in the sample. A binding protein or binding domain (or its fusion protein) can be classified as a "high affinity" binding protein or binding domain (or its fusion protein) or as a "low affinity" binding protein or binding domain (or its fusion protein). A "high affinity" binding protein or binding domain refers to a K a For 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 "Low affinity" binding proteins or binding domains are those with a low affinity for the a Up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5M -1 Alternatively, affinity can be defined as the equilibrium dissociation constant (K d ), the unit is M (for example, 10 -5 M to 10 - 13 M).

[0070] 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 a wild-type (or parent) binding domain. For example, enhanced affinity may be due to a K of the target antigen. a (equilibrium association constant) higher than the K of the wild-type binding domain a , due to the K of the target antigen d (dissociation constant) is less than the K of the wild-type binding domain d , because the dissociation rate (koff) of the target antigen is less than the dissociation rate of the wild-type binding domain, or a combination thereof.

[0071] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, and for determining the affinity of a binding domain or fusion protein, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance. 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 and 5,468,614, or equivalents). Binding and binding affinity can also be assessed using, for example, the fluorescence intensity observed when the binding protein is bound to a labeled HLA peptide complex or a labeled HLA peptide complex multimer (e.g., tetramer).

[0072] 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) can be soluble and can bind to KRAS. d Less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13In 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, such as a protein comprising Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα, or Vα-L-Vβ-Cβ, wherein 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 a linker 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, wherein Vα and Vβ are TCR α and β variable domains, respectively, and L is a linker, such as a linker described herein).

[0073] As used herein, the term "functional avidity" refers to a biological measure or activation threshold of the response of an in vitro immune cell (e.g., T cell, NK cell, NK-T cell) to a given concentration of a ligand, wherein the biological measure 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 make biological (immune) responses to low antigen doses in vitro by, for example, producing cytokines, showing cytotoxic activity or proliferation are considered to have high functional avidity, while T cells with lower functional avidity require a higher amount of antigen before triggering an immune response, similar to high-avidity T cells. It should be understood that functional avidity is different from affinity and avidity. Avidity refers to the strength of any given bond between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to multiple antigens-in this case, the strength of the overall connection is avidity.

[0074] There are many correlations between functional avidity and the effectiveness of the immune response. Some in vitro studies have shown that different T cell functions (e.g., proliferation, cytokine production, etc.) can be triggered at different thresholds (see, for example, Betts et al., Journal of Immunology 172:6407, 2004; Langenkamp et al., Eur. J. Immunol. 32:2046, 2002). Factors affecting functional avidity can include (a) the affinity of TCR to pMHC complex, i.e., the intensity of interaction between TCR and pMHC (Cawthon et al., Journal of Immunology 167:2577, 2001), (b) the expression level of TCR on the host cell, and in some embodiments the expression level of CD4 or CD8 co-receptors and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), as well as the expression level of molecules that weaken T cell function and TCR signaling.

[0075] The concentration of antigen required to induce a half-maximal response (e.g., production of a cytokine or activation marker by host cells; fluorescence intensity upon binding to a labeled peptide:HLA multimer) between baseline and maximal response after a specific exposure time is called the "half-maximal effective concentration" or "EC50." EC50 values ​​are usually expressed as moles (mol / L), but are often converted to logarithmic values ​​as follows – log 10 (EC50). For example, if EC50 is equal to 1 μM (10 -6 M), then log 10 The (EC50) value is -6. Another value used is pEC50, which is defined as the negative logarithm of EC50 (-log 10 (EC50)). In the above example, the pEC50 value equal to an EC50 of 1 μM is 6. In certain embodiments, the functional affinity of the binding proteins of the present disclosure will comprise a measure of the ability of the binding protein to promote T cell activation and / or IFNγ production, which can be measured using assays known in the art and described herein. In certain embodiments, the functional affinity will comprise a measure of the ability of the binding protein to activate host cells (such as T cells) after binding to the antigen.

[0076] The binding proteins disclosed herein can comprise high functional avidity that can, for example, facilitate binding to even lower levels of (e.g., HLA-A * Stimulation of immune cell effector functions (e.g., activation, proliferation, cytokine production and / or cytotoxicity) in response to a KRAS G12 mutant peptide (such as a KRAS G12V mutant peptide of SEQ ID NO: 2 or SEQ ID NO: 3) presented by a KRAS G12 mutant peptide (e.g., a KRAS G12V mutant peptide of SEQ ID NO: 11:01).

[0077] In some embodiments, the binding protein has a log10EC50 for a KRAS G12 (e.g., G12V) mutant peptide 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. or less, about -7.9 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.

[0078] In some embodiments, the host cells disclosed herein comprise a binding protein (e.g., a TCR) that binds to the target antigen of the binding protein (e.g., a KRAS G12 mutant peptide, such as a KRAS G12V mutant peptide, e.g., a peptide present on HLA (e.g., HLA-A)) at an EC50 (e.g., the peptide dose that achieves half-maximal activation of a population of T cells expressing the binding protein). *11:01) complex) binds: less than about 100mM, less than about 10mM, less than about 1mM, less than about 500μM, less than about 100μM, less than about 50μM, less than about 10μM, less than about 5μM, less than about 4μM, less than about 3μM, less than about 2μM, less than about 1μM, less than about 900nM, less than about 800nM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, 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. The EC50 can be determined by an assay that identifies the peptide dose that achieves half-maximal activation of a T cell population, e.g., as reflected by expression of activation markers (e.g., CD137, CD69, granzyme B, CD107a, IFN-γ, TNF-α, IL-12, Nur77, cytokines, interleukins, interferons) by target cells exposed to the mutant peptide in the presence of varying concentrations.

[0079] In some embodiments, the host cells disclosed herein comprise a binding protein (e.g., a TCR) that binds to the target antigen of the binding protein (e.g., a KRAS G12 mutant peptide, such as a KRAS G12V mutant peptide, e.g., a peptide present on HLA (e.g., HLA-A)) at an EC50 (e.g., the peptide dose that achieves half-maximal activation of a population of T cells expressing the binding protein). *11:01) complex) binds to: 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 10 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.

[0080] In some embodiments, the binding protein (e.g., TCR) binds to the target (e.g., a KRAS G12 mutant peptide, such as a KRAS G12V mutant peptide, e.g., a peptide present on HLA (e.g., HLA-A)) with a KD of * 11:01) complex) binds: less than about 100mM, less than about 10mM, less than about 1mM, less than about 500μM, less than about 100μM, less than about 50μM, less than about 10μM, less than about 5μM, less than about 4μM, less than about 3μM, less than about 2μM, less than about 1μM, less than about 900nM, less than about 800nM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, 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.

[0081] Also contemplated are fusion proteins comprising a scTCR or scTv of the present disclosure linked to a constant domain (e.g., a heavy chain constant domain or a combination thereof, such as 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 fragment thereof (e.g., in some embodiments, a fragment that retains 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, such as Fc dimers; see, e.g., Wong et al., J. Immunol. 198:1 Suppl. (2017). Variant Fc polypeptides comprising mutations that enhance, reduce, or eliminate binding to or through, for example, FcRn or other Fc receptors are known and contemplated in the present disclosure.

[0082] In certain embodiments, the binding protein or fusion protein (e.g., TCR, scTCR, CAR) of the present disclosure is expressed by a host cell (e.g., by a T cell, NK cell, or NK-T cell expressing a binding protein or fusion protein heterologously). Such host cells are to KRAS (or NRAS, or HRAS) peptide antigens or KRAS (or NRAS, or HRAS) peptide antigens: The affinity of the HLA complex can be determined by, for example, the following: the host cell is exposed to the peptide or to the peptide: HLA complex (e.g., organized as a tetramer) or exposed to antigen presenting cells (APCs), which present the peptide to the host cell, optionally in the form of a peptide: HLA complex, and then measuring the activity of the host cell, such as cytokine (e.g., IFN-γ; TNFα) production or secretion; host cell signaling or expression of activation components (e.g., CD137 (4-1BB)) is increased; host cell proliferation; or killing APC (e.g., using labeled chromium release assay).

[0083] As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, polynucleotides, for example, fragments thereof produced by polymerase chain reaction (PCR) or in vitro translation, and also refers to fragments produced by any of connection, breakage, endonuclease action or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (for example, α-enantiomeric forms of naturally occurring nucleotides), or a combination of the two. Modified nucleotides can have modifications or replacements of sugar moieties or pyrimidine or purine base moieties. Nucleic acid monomers can be connected by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphothioates, dithiophosphoates, selenophosphoates, diselenphosphoates, thioanilide phosphates, anilide phosphates, aminophosphoesters, etc. Nucleic acid monomers can contain phosphorothioate linkages, phosphorodithioate linkages, or phosphoroselenoate linkages, or any combination thereof. Nucleic acid molecules can be single-stranded or double-stranded.

[0084] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs naturally). 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 nucleic acids can be part of a vector and / or such nucleic acids or polypeptides can be part of a composition (e.g., a cell lysate) and still be isolated because such vectors or compositions are not part of the natural environment of the nucleic acid or polypeptide. In some embodiments, isolated binding proteins, polynucleotides, vectors or host cells are provided. The term "gene" means a segment of DNA involved in producing a polypeptide chain; it includes regions before and after the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons).

[0085] As used herein, the terms "recombinant," "engineered," and "modified" refer to cells, microorganisms, nucleic acid molecules, polypeptides, proteins, plasmids, or vectors that have been modified by the introduction of exogenous nucleic acid molecules, or to cells or microorganisms that have been genetically engineered by human intervention, i.e., modified by the introduction of heterologous nucleic acid molecules, or to cells or microorganisms that have been altered so that the expression of endogenous nucleic acid molecules or genes is controlled, deregulated, or constitutive, wherein such alterations or modifications can be introduced by genetic engineering. Human-induced genetic changes can include, for example, modifications that introduce nucleic acid molecules encoding one or more proteins or enzymes (which can include expression control elements, such as promoters), or additions, deletions, substitutions of other nucleic acid molecules, or other functional disruptions or additions of the genetic material of the cell. Exemplary modifications include those in the coding region of a heterologous or homologous polypeptide from a reference or parent molecule, or a functional fragment thereof.

[0086] 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. A mutation may cause several different types of sequence changes, including substitutions, insertions, or deletions of nucleotides or amino acids. 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.

[0087] "Conservative substitutions" are considered in the art to be substitutions of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97 / 09433, page 10; Lehninger, Biochemistry, 2nd ed.; Worth Publishers, Inc. NY, NY, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, page 8, 1990).

[0088] In certain embodiments, proteins (e.g., binding proteins, immunogenic peptides) according to the present disclosure comprise variant sequences compared to a reference sequence (e.g., variant TCR CDRs (e.g., CDR3β) compared to a reference TCR CDR (e.g., CDR3β) disclosed herein). As used herein, a "variant" amino acid sequence, peptide, or polypeptide may refer to an amino acid sequence (or peptide or polypeptide) having one, two, or three amino acid substitutions, deletions, and / or insertions compared to a reference amino acid sequence. In certain embodiments, the variant amino acid sequence, peptide or polypeptide retains substantially the same function as the reference molecule (e.g., binding specificity and affinity for the peptide:HLA complex); for example, a variant TCR fragment as disclosed herein retains about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or 100% of the antigen binding specificity and affinity compared to a reference TCR binding fragment.

[0089] By “altered domain” or “altered protein” is meant 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% or at least 99.9%) non-identical sequence identity to 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).

[0090] The altered domain or altered protein or derivative can include altered domains or altered proteins or derivatives based on all possible codon selections for the same amino acid and codon selection based on 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), glutamic acid (glu; E); 3) asparagine (asn; N), 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). (See also WO 97 / 09433, page 10, Lehninger, Biochemistry, 2nd ed., Worth Publishing Company, New York, NY, pp. 71-77, 1975; Lewin, Gene IV, Oxford University Press, New York, NY and Cell Press, Cambridge, MA, p. 8, 1990; Creighton, Proteins, WH Freeman and Company, 1984.) In addition, single substitutions, deletions, or additions that change, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are also "conservative substitutions."

[0091] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule. "Transgenic" or "transgenic construct" refers to a construct containing two or more genes that are operably connected in an arrangement not present in nature. The term "operably connected (operably-linked)" (or herein "operably linked (operably linked)") refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, when a promoter can affect the expression of a coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter), the promoter is operably connected to the coding sequence. "Unconnected" means that the related genetic elements are not closely related to each other and the function of one does not affect the other. In some embodiments, the gene present in the transgenic is operably connected to an expression control sequence (e.g., a promoter).

[0092] Constructs (e.g., transgenics) can be present in vectors (e.g., bacterial vectors, viral vectors) or can be integrated into a genome." vector " is a nucleic acid molecule capable of transporting another nucleic acid molecule. A vector can be, for example, a plasmid, a cosmid, a virus, an RNA vector or a linear or circular DNA or RNA molecule that can include a chromosome, a non-chromosomal, semi-synthetic or synthetic nucleic acid molecule. An exemplary vector is a vector capable of autonomous replication (episomal vector) or expression of a nucleic acid molecule (expression vector) connected thereto. The vectors for the compositions and methods of the present disclosure are further described herein.

[0093] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0094] In the context of inserting a nucleic acid molecule into a cell, the term "introduced" 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 incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0095] As used herein, "heterologous" or "exogenous" nucleic acid molecules, constructs or sequences refer to nucleic acid molecules or a portion of nucleic acid molecules that are not natural to the host cell, but can be homologous to nucleic acid molecules or a portion of nucleic acid molecules from the host cell. The source of heterologous or exogenous nucleic acid molecules, constructs or sequences can be from different genera or species. In certain embodiments, heterologous or exogenous nucleic acid molecules are added (i.e., not endogenous or natural) to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, etc., wherein the added molecules can be integrated into the host genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can exist in multiple copies. In addition, "heterologous" refers to non-natural enzymes, proteins or other activities encoded by exogenous nucleic acid molecules introduced into the host cell, even if the host cell encodes homologous proteins or activities. In addition, cells comprising "modified" or "heterologous" polynucleotides or protein-binding proteins include the offspring of the cells, whether or not the offspring itself is transduced, transfected or otherwise manipulated or changed.

[0096] As described herein, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as independent nucleic acid molecules, as multiple individually controlled genes, as polycistronic nucleic acid molecules, 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, as disclosed herein, also encoding a CD8 co-receptor polypeptide comprising an α chain, a β chain, or a portion thereof, such as an extracellular portion capable of binding to MHC. When two or more exogenous nucleic acid molecules are introduced into a host cell, it should be understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), introduced on independent vectors, 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 referred to refers to the number of encoding nucleic acid molecules or the number of protein activities, and does not necessarily refer to the number of independent nucleic acid molecules introduced into the host cell.

[0097] 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, reorganized, duplicated, or otherwise altered compared to the parent gene, protein, or activity is still considered endogenous or native to the particular host cell. For example, endogenous control sequences (e.g., promoters, translation attenuation sequences) 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 the normal expression or regulation in the parent cell.

[0098] The term "homolog" or "homolog" refers to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene and can optionally have altered expression levels, a different sequence, an altered activity, or any combination thereof.

[0099] As used herein, " sequence identity " refers to after comparing sequences and introducing room (if necessary) to realize maximum sequence identity percentage and not considering any conservative substitution as a part for sequence identity, the percentage of amino acid residue or core base (respectively) identical with the amino acid residue in the reference sequence or core base in a sequence. Percent sequence identity values ​​can be generated using the NCBI BLAST 2.0 software defined in, for example, Altschul et al. (1997), " Nucleic Acids Research (Nucl.Acids Res.) " 25:3389-3402, wherein parameters are set to default values. Additionally or alternatively, for example, by using a computer program designed for this purpose (such as global or local alignment algorithm) to compare two sequences, the degree of sequence identity between two sequences can be determined. 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. Global alignment algorithms, such as Needleman and Wunsch algorithms, can be used for comparing the entire length of two sequences, maximizing the quantity of coupling and minimizing the quantity in the room. Default settings can be used.

[0100] To generate a similarity score for two amino acid sequences, some non-identical amino acids (e.g., conservative amino acid substitutions, amino acids with similar physicochemical properties, and / or amino acids that exhibit frequent substitutions among orthologs, homologs, or paralogs) can be assigned positive scores using a scoring matrix, non-limiting examples of which include PAM30, PAM70, PAM250, BLOSUM45, BLOSUM50, BLOUM62, BLOSUM80, and BLOSUM90.

[0101] 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%, and 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 as defined or referenced herein, or hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65°C-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Variants of the nucleic acid molecules retain the ability to encode a binding protein or its binding domain having the functions described herein, such as binding to a target molecule.

[0102] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs naturally). 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 can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" means a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

[0103] In some contexts, as used herein, the term "variant" refers to at least one fragment of the full-length sequence referred to, more specifically, to one or more amino acids or nucleic acid sequences truncated at one or both ends relative to the full-length sequence. Such fragments include or encode peptides of 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 total length of the 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.

[0104] In some embodiments, the term "variant" refers not only to at least one fragment, but also 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, wherein amino acids other than those essential for the biological activity or 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 such that the biological activity of the polypeptide is retained. The prior art includes various methods that can be used to compare two given nucleic acid or amino acid sequences and to 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 (Larkin, MA et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) can be used with default settings.

[0105] In certain embodiments, variants may further include chemical modifications, such as isotopic labeling or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, etc. Methods for modifying polypeptides are known and are generally employed so as not to eliminate or substantially reduce a desired activity of the polypeptide.

[0106] In one embodiment, the term "variant" of a nucleic acid molecule includes nucleic acids whose complementary chains hybridize, for example, to a reference or wild-type nucleic acid under stringent conditions. The stringency of the hybridization reaction can be easily determined by one of ordinary skill in the art and is typically an empirical calculation dependent on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes do not. Hybridization generally depends on the ability of denatured DNA to reanneal to the complementary chain (present in an environment below its melting temperature): the higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative humidity that can be used. Therefore, higher relative humidity tends to make the reaction conditions more stringent, while lower temperatures do not. For further details and explanations on the stringency of the hybridization reaction, see Ausubel, FM (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Alternatively, one skilled in the art may follow the instructions given in the manual "The DIG System Users Guide for Filter Hybridization" by Boehringer Mannheim GmbH (1993), "Boehringer Mannheim GmbH, Mannheim, Germany," and the instructions given in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, KH (1991) "International Journal of Systematic Bacteriology," 41: 255-260, regarding how to identify DNA sequences by hybridization. In one embodiment, stringent conditions are applied to any hybridization, i.e., hybridization will only occur if the probe is 70% or more identical to the target sequence. Probes with a lower degree of identity to the target sequence can hybridize, but such hybrids are unstable and will be removed during wash steps under stringent conditions, for example, by reducing the salt concentration to 2×SSC, or optionally and subsequently to 0.5×SSC, while the temperature is, for example, about 50° C.–68° C., about 52° C.–68° C., about 54° C.–68° C., about 56° C.–68° C., about 58° C.–68° C., about 60° C.–68° C., about 62° C.–68° C., about 64° C.–68° C., or about 66° C.–68° C. In one embodiment, the temperature is about 64° C.–68° C. or about 66° C.–68° C. The salt concentration can be adjusted to 0.2×SSC or even 0.1×SSC.Nucleic acid sequences can be isolated that have a degree of identity 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% to a reference or wild-type sequence. In one embodiment, as used herein, the term variant of a nucleic acid sequence refers to any nucleic acid sequence that encodes the same amino acid sequence as a reference nucleic acid sequence and variants thereof, consistent with the degeneracy of the genetic code.

[0107] "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 in some contexts slightly different 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 activity level of the parent polypeptide. In other words, when a functional variant of a polypeptide or encoded polypeptide of the present disclosure exhibits no more than 50% reduction in performance compared to the parent or reference polypeptide in a selected assay, such as for measuring binding affinity (e.g., measuring association (Ka) or dissociation (KD) constants), the polypeptide or encoded polypeptide is a polypeptide or polynucleotide that is structurally similar or substantially similar to a parent or reference compound of the present disclosure, but in some contexts slightly different 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), 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 activity level of the parent polypeptide. or tetramer staining), affinity or activation of host cells, the functional variant has "similar binding", "similar affinity" or "similar activity". As used herein, "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that contains only a domain, motif, part or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, part 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 activity level of the parent polypeptide, or provides a biological benefit (e.g., effector function).

[0108] When a "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure exhibits no more than a 50% reduction in performance compared to a parent or reference polypeptide in a selected assay (preferably no more than 20% or 10%, or no more than a log difference in affinity compared to a parent or reference), such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release), the functional portion or fragment has "similar binding" or "similar activity." Functional variants of the specifically disclosed binding proteins and polynucleotides are contemplated.

[0109] By an “altered domain” or “altered protein” is meant 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% or at least 99.9%) non-identical sequence identity to 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).

[0110] Binding protein

[0111] In one aspect, the present disclosure provides a binding protein comprising a T cell receptor (TCR) α chain variable (Vα) domain and a TCR β chain variable (Vβ) domain, 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 shown 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 presently disclosed embodiments, the binding protein can be heterologously expressed by a human immune system cell (eg, a T cell).

[0112] In certain embodiments, the Vα domain and / or the Vβ domain are each independently human, humanized or chimeric, and 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 therefrom derived from human subjects, for example, from isolated T cells from human subjects or their populations sequenced. The TCRVα domains, Vβ domains, and CDRs therefrom isolated from human subjects can have advantageous properties over variable domains and CDRs from other sources, such as transgenic mice for single human HLA alleles. For example, Vα domains, Vβ domains, and CDRs derived from human subjects can be subjected to negative thymic selection in vivo for a substantially full set of human polypeptides presented by a full set of human HLA molecules, which can reduce the possibility of cross-reactions of the binding protein to other human self-antigens.

[0113] In some embodiments, the binding proteins disclosed herein are substantially unresponsive to the human proteome presented by one or more HLA alleles disclosed herein (e.g., one or any combination of the HLA alleles in Table 3). Reactivity can be determined by any suitable method, such as the methods disclosed in Examples 5-7 and 13 of the present application. In some embodiments, no significant response is observed or predicted by binding of T cells transduced with the binding protein to the human proteome presented by one or more HLA alleles at a peptide concentration 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 unresponsive to a peptide:HLA complex, wherein the peptide comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs.: 118-148. In some embodiments, the HLA comprises HLA-A * 11. In another embodiment, HLA comprises HLA-A * 11:01.

[0114] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells (e.g., identified in T cells) of a subject (e.g., a human subject) suffering from a disease (e.g., cancer). In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a human subject suffering from a cancer disclosed herein. In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a subject (e.g., a human subject) suffering from a disease associated with a KRAS G12 mutation (e.g., KRASG12V or G12D mutation). In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of a subject (e.g., a human subject) having a cell comprising a KRAS G12 mutation (e.g., KRAS G12V or G12D mutation).

[0115] In some embodiments, the binding protein comprises one or more variable domains or one or more CDRs derived from T cells of healthy subjects (e.g., healthy human subjects). In some embodiments, the healthy subjects lack a specific pathological diagnosis (e.g., a disease diagnosis, such as a cancer diagnosis). In some embodiments, the healthy subjects lack a specific pathological diagnosis but have a different pathological diagnosis (e.g., lack a cancer diagnosis but have a diagnosis of hypertension or type II diabetes).

[0116] The presently disclosed binding proteins can be heterologously expressed by host cells, such as human immune cells, such as T cells. In addition, expression of the presently disclosed binding proteins can confer advantageous properties on the host cells; for example, binding specificity for the disclosed Ras antigen:HLA complex, and improved activation, proliferation, or killing activity in the presence of Ras antigen:HLA presenting tumor cells, etc.

[0117] For example, in certain embodiments, when the binding protein is expressed by immune cells (e.g., human T cells, optionally CD8+ and / or CD4+ T cells, NK cells or NK-T cells), the immune cells are able to specifically kill HLA-A cells expressing a peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO.: 2 or 3. * 11:01 + Tumor cells. For example, A bioimaging platform (Essen Bioscience) can determine target cell killing. In certain embodiments, this platform uses activated caspase and labeled (e.g., RapidRed or NucRed) tumor cell signals, where overlap is measured and increased overlap area equates to tumor cell death by apoptosis. Killing can also be determined using a 4-hour assay where target cells are loaded with labeled chromium ( 51 Cr) and measure the supernatant 51 Cr, for example, after 4 hours of incubation with immune cells expressing binding proteins of the present 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.

[0118] In any of the presently disclosed embodiments, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), when there is an HLA-A11:01 expressing the peptide + In the presence of tumor cells, optionally in the further presence of exogenous IFN-γ, the immune cells have elevated expression of Nur77, the peptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 2 or 3, wherein Nur77 expression is elevated compared to: (i) Nur77 expression by a reference immune cell that does not express the binding protein (i.e., is of the same cell type as the immune cell that expresses the binding protein and is otherwise at least substantially identical or functionally equivalent thereto in terms of phenotype and / or genotype) when the reference immune cell is present in the tumor cells; and / or (ii) Nur77 expression by an immune cell that expresses the binding protein when tumor cells are not present and / or in the absence of an antigen-presenting cell that expresses the peptide:HLA complex, wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.: 2 or 3, and wherein the HLA is optionally HLA-A * 11: 01. For example, Nur77 expression can be determined using a transgenic expression construct comprising a Nur77 locus operably linked to a sequence encoding a reporter gene construct; for example, dTomato (see Ahsouri and Weiss, J. Immunol. 198(2):657-668 (2017)).

[0119] In any of the presently disclosed embodiments, when the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell), the binding protein is expressed by an immune cell (e.g., a human T cell, optionally a CD8+ and / or CD4+ T cell, a NK cell, or a NK-T cell) in the presence of an HLA-A * 02 + The immune cells have elevated expression of CD137 (also known as 4-1BB) in the presence of tumor cells, optionally in the further presence of exogenous IFN-γ, the peptide comprising, consisting essentially of, or consisting of the amino acid sequence set forth in SEQ ID NO.: 2 or 3, wherein CD137 expression is elevated compared to: (i) CD137 expression of a reference immune cell that does not express the binding protein when the reference immune cell is present in the presence of the tumor cell; and / or (ii) CD137 expression of an immune cell that expresses the binding protein when the tumor cell is not present and / or in the absence of an antigen-presenting cell that expresses the peptide:HLA complex, wherein the peptide comprises, consists essentially of, or consists of the amino acid sequence set forth in SEQ ID NO.: 2 or 3, and wherein the HLA is optionally HLA-A * 11:01. CD137 expression can be determined using, for example, flow cytometry using a labeled anti-CD137 antibody. In certain embodiments, CD137 is measured after 16 hours of an assay in which immune cells are co-incubated with or stimulated with the peptide or target cells expressing the peptide.

[0120] In any of the presently disclosed embodiments: (i) the binding protein is encoded by a polynucleotide heterologous to the immune cell; (ii) the immune cell comprises human CD8 + T cells, human CD4+ T cells or both; (iii) tumor cells expressing a peptide comprising or consisting of the amino acid sequence shown 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.

[0121] In certain embodiments, the binding protein is capable of binding to the peptide:HLA complex independently of CD8 or in the absence of CD8. CD8-independent binding can be achieved by binding to CD8-negative cells (e.g., CD4 +The binding protein is expressed in T cells, Jurkat cells, etc. and the binding of the cells to the target is identified. In some embodiments, a binding protein is provided that comprises: (a) a T cell receptor (TCR) alpha 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 beta chain variable (Vβ) domain comprising the CDR3β 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, 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 wherein the HLA comprises HLA-A * 11. In certain embodiments, the HLA comprises HLA-A * 11:01. The binding protein may comprise a Vα domain and a Vβ domain.

[0122] The Va domain and / or the Vβ domain may be human, humanized or chimeric, and is preferably human.

[0123] In certain embodiments, the binding protein comprises the CDR3α and CDR3β amino acid sequences set forth in: (i) SEQ ID NOs.: 17 and 27, respectively, or variants thereof having one, two, or three optionally conservative amino acid substitutions; (ii) SEQ ID NOs.: 16 and 26, respectively, or variants thereof having one, two, or three optionally conservative amino acid substitutions; (iii) SEQ ID NOs.: 53 and 43, respectively, or variants thereof having one, two, or three optionally conservative amino acid substitutions; or (iv) SEQ ID NOs.: 52 and 42, respectively, or variants thereof having one, two, or three optionally conservative amino acid substitutions. In certain embodiments, the binding protein comprises the CDR3α and CDR3β amino acid sequences set forth in: (i) SEQ ID NOs.: 17 and 27, respectively; (ii) SEQ ID NOs.: 16 and 26, respectively; (iii) SEQ ID NOs.: 53 and 43, respectively; or (iv) SEQ ID NOs.: 52 and 42, respectively.

[0124] In some embodiments, the binding protein further comprises: (i) in the Vα domain, the CDR1α amino acid sequence shown in SEQ ID NO.: 14 or 40, or a variant thereof having one or two optionally conservative amino acid substitutions; (ii) in the Vα domain, the CDR2α amino acid sequence shown in SEQ ID NO.: 15 or 41, or a variant thereof having one or two optionally conservative amino acid substitutions; (iii) in the Vβ domain, the CDR1β amino acid sequence shown in SEQ ID NO.: 24 or 50, or a variant thereof having one or two optionally conservative amino acid substitutions; (iv) in the Vβ domain, the CDR2β amino acid sequence shown in SEQ ID NO.: 25 or 51, or a variant thereof having one or two optionally conservative amino acid substitutions; or any combination of (v)(i)-(iv).

[0125] In some embodiments, the binding protein further comprises: (i) in the Vα domain, the CDR1α amino acid sequence shown in SEQ ID NO.: 14 or 40; (ii) in the Vα domain, the CDR2α amino acid sequence shown in SEQ ID NO.: 15 or 41; (iii) in the Vβ domain, the CDR1β amino acid sequence shown in SEQ ID NO.: 24 or 50; (iv) in the Vβ domain, the CDR2β amino acid sequence shown in SEQ ID NO.: 25 or 51; or any combination of (v)(i)-(iv).

[0126] In some embodiments, the binding protein further comprises: (i) in the Vα domain, the CDR1α amino acid sequence shown in SEQ ID NO.: 14 or 40; (ii) in the Vα domain, the CDR2α amino acid sequence shown in SEQ ID NO.: 15 or 41; (iii) in the Vβ domain, the CDR1β amino acid sequence shown in SEQ ID NO.: 24 or 50; and (iv) in the Vβ domain, the CDR2β amino acid sequence shown in SEQ ID NO.: 25 or 51.

[0127] In certain embodiments, the binding protein comprises 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.

[0128] In other embodiments, the binding protein comprises 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 52, respectively.

[0129] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as 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 as 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 as 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 as identified by the Kabat method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the Kabat method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β as 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 as identified by the Kabat method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the Kabat method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β as identified by the Kabat method from the variable domain sequence of SEQ ID NO: 49.

[0130] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as 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 as 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 as 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 as identified by the Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β as 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 as identified by the Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β as identified by the Chothia method from the variable domain sequence of SEQ ID NO: 49.

[0131] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as identified by the EU 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 as identified by the EU 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 as identified by the EU 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 as identified by the EU method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β as identified by the EU method from the variable domain sequence of SEQ ID NO: 23. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α as identified by the EU method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β as identified by the EU 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 as identified by the EU method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β as identified by the EU method from the variable domain sequence of SEQ ID NO: 49. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α as identified by the EU method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β as identified by the EU method from the variable domain sequence of SEQ ID NO: 49.

[0132] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as identified by the IMGT method or numbering scheme (including IMGT and / or IMGT junction of 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 as 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 as 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 as identified by the IMGT method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the IMGT method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β as 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 as identified by the IMGT method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the IMGT method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β as identified by the IMGT method from the variable domain sequence of SEQ ID NO: 49.

[0133] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as identified by the IMGT method or numbering scheme (including IMGT and / or IMGT junction of CDR3) from the amino acid sequence shown in SEQ ID NO.: 20, the amino acid sequence shown in SEQ ID NO.: 30, the amino acid sequence shown in SEQ ID NO.: 155, or a combination thereof. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and / or CDR3α as identified by the IMGT method from the amino acid sequence shown in SEQ ID NO.: 20. In some embodiments, the binding proteins disclosed herein comprise CDR1β, CDR2β, and / or CDR3β as identified by the IMGT method from the amino acid sequence shown in SEQ ID NO.: 30 or SEQ ID NO.: 155. In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, and CDR3α as identified by the IMGT method from the amino acid sequence shown in SEQ ID NO.: 20, and CDR1β, CDR2β, and CDR3β as identified by the IMGT method from the amino acid sequence shown in SEQ ID NO.: 30 or SEQ ID NO.: 155.

[0134] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as identified by the enhanced 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 as identified by the enhanced 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 as identified by the enhanced 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 as identified by the enhanced Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β as identified by the enhanced 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 as identified by the enhanced Chothia method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β as identified by the enhanced 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 as identified by the enhanced Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β as identified by the enhanced 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 as identified by the enhanced Chothia method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β as identified by the enhanced Chothia method from the variable domain sequence of SEQ ID NO: 49.

[0135] In some embodiments, the binding proteins disclosed herein comprise CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and / or CDR3β as 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 as 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 as 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 as identified by the Aho method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the Aho method from the variable domain sequence of SEQ ID NO: 13, and CDR1β, CDR2β, and CDR3β as 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 as identified by the Aho method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and / or CDR3β as 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 as identified by the Aho method from the variable domain sequence of SEQ ID NO: 39, and CDR1β, CDR2β, and CDR3β as identified by the Aho method from the variable domain sequence of SEQ ID NO: 49.

[0136] In some embodiments, the binding protein comprises a CDR1α comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 14 or 40, or a CDR1α sequence as identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0137] In some embodiments, the binding protein comprises a CDR2α comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 15 or 41, or a CDR2α sequence as identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0138] In some embodiments, the binding protein comprises a CDR3α comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 16, 17, 42, or 43, or a CDR3α sequence as identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0139] In some embodiments, the binding protein comprises a CDR1β comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 24 or 50, or a CDR1β sequence as identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho methods.

[0140] In some embodiments, the binding protein comprises a CDR2β comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 25 or 51, or a CDR2β sequence as identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, Enhanced Chothia, or Aho methods.

[0141] In some embodiments, the binding protein comprises a CDR3β comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 26, 27, 52, or 53, or a CDR3β sequence as identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0142] In some embodiments, the binding protein comprises a CDR1α comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 14 or 40, or a CDR1α sequence identified from the variable domain of SEQ ID NO: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0143] In some embodiments, the binding protein comprises a CDR2α comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 15 or 41, or a CDR2α sequence identified from the variable domain of SEQ ID NO: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0144] In some embodiments, the binding protein comprises a CDR3α comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 16, 17, 42, or 43, or a CDR3α sequence identified from the variable domain of SEQ ID NO: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0145] In some embodiments, the binding protein comprises a CDR1β comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 24 or 50, or a CDR1β sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0146] In some embodiments, the binding protein comprises a CDR2β comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 25 or 51, or a CDR2β sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0147] In some embodiments, the binding protein comprises a CDR3β comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 26, 27, 52, or 53, or a CDR3β sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0148] In some embodiments, the binding protein comprises a CDR1α comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 14 or 40, or a CDR1α sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertions and / or deletions can be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0149] In some embodiments, the binding protein comprises a CDR2α comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 15 or 41, or a CDR2α sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertions and / or deletions can be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0150] In some embodiments, the binding protein comprises a CDR3α comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 16, 17, 42, or 43, or a CDR3α sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertions and / or deletions can be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0151] In some embodiments, the binding protein comprises a CDR1β comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 24 or 50, or a CDR1β sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions may be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0152] In some embodiments, the binding protein comprises a CDR2β comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 25 or 51, or a CDR2β sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions can be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0153] In some embodiments, the binding protein comprises a CDR3β comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 26, 27, 52, or 53, or a CDR3β sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions can be at the N-terminus of the CDR, at the C-terminus of the CDR, within the amino acid sequence of the CDR, or a combination thereof.

[0154] The binding proteins disclosed herein may comprise one or more framework regions (FRs). For example, the binding protein may comprise one variable domain comprising three CDRs and four FRs, or two variable domains each comprising three CDRs and four FRs. Illustrative FR amino acid sequences are provided by SEQ ID NOs: 91-117 and 153. The framework regions used in the binding protein may be mammalian framework regions. The framework regions used in the binding protein may be human framework regions. The framework regions used in the binding protein may be engineered framework regions.

[0155] The binding protein may comprise FR1, FR2, FR3, and / or FR4 disclosed herein. In some embodiments, the binding protein comprises Vα, wherein Vα comprises FR1, FR2, FR3, and FR4, wherein FR1 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, FR2 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, FR3 comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 93, 95, 105, 107, or a variant thereof, FR4 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 Vα, wherein Vα comprises the amino acid sequence set forth in SEQ ID NO.: 115. In some embodiments, the binding protein comprises Vα comprising the amino acid sequence set forth in SEQ ID NO.:91.

[0156] In some embodiments, the binding protein comprises a Vβ comprising FR1, FR2, FR3, and FR4, wherein FR1 comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 97, 109, and 153, or a variant thereof, FR2 comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 98, 110, or a variant thereof, FR3 comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 99, 101, 111, 113, or a variant thereof, and FR4 comprises, consists essentially of, or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 100, 102, 112, 114, 116, and 117, or a variant thereof.

[0157] 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.

[0158] 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.

[0159] In some embodiments, the binding protein comprises a Va 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.

[0160] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as identified by the EU methodology 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 EU methodology from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0161] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as 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 as identified by the IMGT method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0162] In some embodiments, the binding protein comprises a Vα domain comprising FR1, FR2, FR3, and FR4 as identified by the enhanced 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 enhanced Chothia method from the variable domain sequence of SEQ ID NO: 23 or SEQ ID NO: 49.

[0163] 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.

[0164] In some embodiments, the binding protein comprises a Vα domain comprising a FR1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 91, 103, or 115, or a FR1 sequence identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0165] In some embodiments, the binding protein comprises a Vα domain comprising a FR2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 92 or 104, or a FR2 sequence as identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0166] In some embodiments, the binding protein comprises a Va domain comprising a FR3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 93, 95, 105, or 107, or a FR3 sequence identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0167] In some embodiments, the binding protein comprises a Va domain comprising a FR4 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 94, 96, 106, or 108, or a FR4 sequence identified from the variable domain of SEQ ID NO: 13 or 39 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0168] In some embodiments, the binding protein comprises a Vβ domain comprising a FR1 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 97 or 109, or a FR1 sequence identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0169] In some embodiments, the binding protein comprises a Vβ domain comprising a FR2 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 98 or 110, or a FR2 sequence as identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0170] In some embodiments, the binding protein comprises a Vβ domain comprising a FR3 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 99, 101, 111, or 113, or a FR3 sequence identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0171] In some embodiments, the binding protein comprises a Vβ domain comprising a FR4 comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 100, 102, 112, 114, 116, or 117, or a FR4 sequence identified from the variable domain of SEQ ID NO: 23 or 49 by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods.

[0172] In some embodiments, the binding protein comprises a Vα domain comprising a FR1 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NOs: 91, 103, or 115, or a FR1 sequence identified from the variable domain of SEQ ID NOs: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0173] In some embodiments, the binding protein comprises a Vα domain comprising a FR2 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 92 or 104, or a FR2 sequence identified from the variable domain of SEQ ID NO: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0174] In some embodiments, the binding protein comprises a Vα domain comprising a FR3 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 93, 95, 105, or 107, or a FR3 sequence identified from the variable domain of SEQ ID NO: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0175] In some embodiments, the binding protein comprises a Vα domain comprising a FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 94, 96, 106, or 108, or an FR4 sequence identified from the variable domain of SEQ ID NO: 13 or 39 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0176] In some embodiments, the binding protein comprises a Vβ domain comprising a FR1 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 97 or 109 or 153, or a FR1 sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions can be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0177] In some embodiments, the binding protein comprises a Vβ domain comprising a FR2 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 98 or 110, or a FR2 sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0178] In some embodiments, the binding protein comprises a Vβ domain comprising a FR3 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 99, 101, 111, or 113, or a FR3 sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions can be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions can be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0179] In some embodiments, the binding protein comprises a Vβ domain comprising a FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 100, 102, 112, 114, 116, or 117, or an FR4 sequence identified from the variable domain of SEQ ID NO: 23 or 49 as determined by Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods. The substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The substitutions may be conservative, non-conservative, or a combination thereof. In some embodiments, the substitutions are conservative.

[0180] In some embodiments, the binding protein comprises a Va domain comprising a FR1 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NOs: 91, 103, or 115, or a FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NOs: 13 or 39. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0181] In some embodiments, the binding protein comprises a Va domain comprising a FR2 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 92 or 104, or a FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0182] In some embodiments, the binding protein comprises a Va domain comprising a FR3 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 93, 95, 105, or 107, or a FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0183] In some embodiments, the binding protein comprises a Va domain comprising a FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 94, 96, 106, or 108, or a FR4 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 13 or 39. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0184] In some embodiments, the binding protein comprises a Vβ domain comprising a FR1 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 97 or 109 or 153, or a FR1 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0185] In some embodiments, the binding protein comprises a Vβ domain comprising a FR2 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 98 or 110, or a FR2 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0186] In some embodiments, the binding protein comprises a Vβ domain comprising a FR3 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 99, 101, 111, or 113, or a FR3 sequence as identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0187] In some embodiments, the binding protein comprises a Vβ domain comprising a FR4 comprising at most one, at most two, at most three, at most four, at most five, or at most six amino acid insertions and / or deletions relative to the amino acid sequence of SEQ ID NO: 100, 102, 112, 114, 116, or 117, or a FR4 sequence identified by the Kabat, Chothia, EU, IMGT, enhanced Chothia, or Aho methods from the variable domain of SEQ ID NO: 23 or 49. The insertions and / or deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof.

[0188] The binding protein may 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.

[0189] 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 shown in SEQ ID NO.: 13 or 39; and / or (ii) the Vβ domain comprises 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 shown in SEQ ID NO.: 13 or 39; NO.: 23 or 154 or 49 having, consisting essentially of, or consisting 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.

[0190] 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 shown in SEQ ID NO.: 13, and the Vβ domain comprises 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 shown in SEQ ID NO.: 13. NO.: 23 or 154 having, consisting essentially of, or consisting 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.

[0191] In some embodiments, the Va domain comprises, consists essentially of, or consists of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity or sequence similarity to the amino acid sequence shown in SEQ ID NO.:39, 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 shown in SEQ ID NO.:49.

[0192] 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, and the TCR β chain comprises amino acids 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 of the amino acid sequence set forth in SEQ ID NO.: 13, and the Vβ domain consists 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 of the amino acid sequence set forth in SEQ ID NO.: 23.

[0193] In some embodiments, a binding protein is provided that comprises a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises the amino acid sequence shown in SEQ ID NO.: 13, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO.: 23 or 154.

[0194] In some embodiments, a binding protein is provided that is capable of binding to a peptide:HLA complex, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NO.: 2 or SEQ ID NO.: 3, and wherein the HLA is optionally HLA-A. * 11, 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 can be or comprise a TCR α chain and / or the second polypeptide can be or comprise a TCR β chain. In some embodiments, the first polypeptide is or comprises a TCR α chain and / or the second polypeptide is or comprises a TCR β chain.

[0195] 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 of the amino acid sequence set forth in SEQ ID NO.: 39, and the Vβ domain consists 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.

[0196] In some embodiments, a binding protein is provided that comprises a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises the amino acid sequence shown in SEQ ID NO.: 13, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO.: 23 or 154.

[0197] In some embodiments, a binding protein is provided that comprises a TCR α chain and a TCR β chain, wherein the TCR α chain comprises the amino acid sequence shown in SEQ ID NO.: 20, and the TCR β chain comprises the amino acid sequence shown in SEQ ID NO.: 30.

[0198] In some embodiments, a binding protein is provided that comprises a TCRα chain and a TCRβ chain, wherein the TCRα chain comprises the amino acid sequence shown in SEQ ID NO.:20, and the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO.:155.

[0199] In some embodiments, a binding protein is provided that is capable of binding to a peptide:HLA complex, wherein the peptide comprises, consists essentially of, or consists of SEQ ID NO.: 2 or SEQ ID NO.: 3, and wherein the HLA is optionally HLA-A. * 11, 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 can be or comprise a TCR α chain and / or the second polypeptide can be or comprise a TCR β chain. In some embodiments, the first polypeptide is or comprises a TCR α chain and / or the second polypeptide is or comprises a TCR β chain.

[0200] 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.

[0201] For example, the variable domain can comprise 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 insertions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0202] In some embodiments, the variable domain 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 insertions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0203] In some embodiments, the variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid insertions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0204] The one or more insertions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions may be continuous, non-contiguous, or a combination thereof.

[0205] 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.

[0206] In some embodiments, the variable domain 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 deletions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0207] In some embodiments, the variable domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions relative to any one of SEQ ID NOs: 13, 23, 39, and 49.

[0208] The one or more deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions may be continuous, non-continuous, or a combination thereof.

[0209] 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.

[0210] In some embodiments, the variable domain 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: 13, 23, 39, and 49.

[0211] 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.

[0212] The one or more substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions may be continuous, non-continuous, or a combination thereof.

[0213] The binding protein may further include a TCR α chain constant domain (Cα) and / or a TCR β chain constant domain (Cβ). The TCR α chain constant domain (Cα) and / or the TCR β chain constant domain (Cβ) may be human. The TCR α chain constant domain (Cα) and / or the TCR β chain constant domain (Cβ) may be mammalian. The TCR α chain constant domain (Cα) and / or the TCR β chain constant domain (Cβ) may be an engineered variant of a mammalian (e.g., human) constant domain. In some embodiments, Cα is an engineered variant of human Cα and / or Cβ is an engineered variant of human Cβ. In some embodiments, Cα is an engineered variant of human Cα and Cβ is an engineered variant of human Cβ.

[0214] In some embodiments, Ca comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence set forth in any one of SEQ ID NOs.: 18, 19, 44, 45, and 69.

[0215] In some embodiments, the Cβ comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence set forth in any one of SEQ ID NOs.: 28, 29, 54, 55, and 70-73.

[0216] In certain embodiments, Cα and Cβ comprise or consist of an amino acid sequence that is 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% identical to the amino acid sequence set forth in: (i) SEQ ID NOs.: 18 and 28, respectively; (ii) SEQ ID NOs.: 19 and 29, respectively; (iii) SEQ ID NOs.: 44 and 54, respectively; or (iv) SEQ ID NOs.: 45 and 55, respectively.

[0217] The binding protein may comprise (i) an extracellular domain of a TCR α chain, a TCR β chain, a TCR γ chain, or a TCR δ chain; (ii) a transmembrane domain of a TCR α chain, a TCR β chain, a TCR γ chain, or a TCR δ chain; and / or (iii) a cytoplasmic domain of a TCR α chain, a TCR β chain, a TCR γ chain, or a TCR δ chain. The binding protein may comprise a full-length or substantially full-length TCR α chain, a TCR β chain, a TCR γ chain, and / or a TCR δ chain.

[0218] 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 comprise or consist of an amino acid sequence that is 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% identical to the amino acid sequence set forth in: (i) SEQ ID NOs.: 12 and 22, respectively; (ii) SEQ ID NOs.: 20 and 30, respectively; (iii) SEQ ID NOs.: 12 and 30, respectively; (iv) SEQ ID NOs.: 20 and 22, respectively; (v) SEQ ID NOs.: 38 and 48, respectively; (vi) SEQ ID NOs.: 46 and 56, respectively; (vii) SEQ ID NOs.: 38 and 56, respectively; or (viii) SEQ ID NOs.: 5 NO.:46 and 48.

[0219] 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, wherein the first polypeptide and the second polypeptide can associate to form a polypeptide dimer.

[0220] 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.

[0221] For example, the binding protein can comprise 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 insertions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0222] 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 insertions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0223] 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 insertions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0224] The one or more insertions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The one or more insertions may be continuous, non-contiguous, or a combination thereof.

[0225] 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.

[0226] 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 deletions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0227] 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 deletions relative to any one of SEQ ID NOs: 12, 18-22, 28-30, 38, 44-46, 48, 54-56, and 69.

[0228] The one or more deletions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The one or more deletions may be continuous, non-continuous, or a combination thereof.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] The one or more substitutions may be at the N-terminus, at the C-terminus, within the amino acid sequence, or a combination thereof. The one or more substitutions may be continuous, non-continuous, or a combination thereof.

[0233] In any embodiment of the presently disclosed embodiment, binding protein can include TCR, single-chain TCR (scTCR), scTv or chimeric antigen receptor (CAR). The method for producing engineered TCR is described in, for example, Bowerman et al., "Molecular Immunology (Mol.Immunol.)", 46 (15): In 3000 (2009), the technology of the document is incorporated herein by reference. The method for preparing CAR is known in the art and is described in, for example, U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT discloses No. WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al., 2007, "Clinical Cancer Research (Clin.Cancer Res.)" 13: In 5426, the technology of the document is incorporated herein by reference. In some embodiments, the binding protein comprises a soluble TCR, optionally fused to a binding domain (e.g., scFv) specific for the CD3 protein. See Elie Dolgin, Nature Biotechnology 40:441-449 (2022).

[0234] In any of the presently disclosed embodiments, the polynucleotide encoding the binding protein may further comprise: (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor alpha chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor alpha chain; (ii) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor beta chain, wherein, optionally, the encoded polypeptide is or comprises a 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 a CD8 co-receptor protein or a portion thereof that functionally binds to an HLA molecule can increase host cell (e.g., immune cell, such as a T cell, optionally CD4 T cells) expression compared to expression of the binding protein alone. + It should be understood that the polynucleotide encoding the binding protein and the polynucleotide encoding the CD8 co-receptor polypeptide can be present on a single nucleic acid molecule (e.g., in the same expression vector), or can be present on separate nucleic acid molecules in the host cell.

[0235] In any of the presently disclosed embodiments, the CD8 co-receptor α chain can comprise, consist essentially of, or consist of SEQ ID NO.: 87, or SEQ ID NO.: 87 with the signal peptide 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 α 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 with the signal peptide removed.

[0236] In any of the presently disclosed embodiments, the CD8 co-receptor β chain can comprise, consist essentially of, or consist of SEQ ID NO.: 89, or SEQ ID NO.: 89 with the signal peptide 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 β 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 with the signal peptide removed.

[0237] In certain additional embodiments, the polynucleotide comprises: (a) said polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; (b) said polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain; and (c) a polynucleotide encoding a self-cleaving peptide, said polynucleotide being positioned between said polynucleotide of (a) and said polynucleotide of (b). In additional embodiments, the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide and being positioned between: (1) said polynucleotide encoding a binding protein and said polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; and / or (2) said polynucleotide encoding a binding protein and said polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain.

[0238] In still further embodiments, the polynucleotide may comprise 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)-(pn BP)-(pnSCP2)-(pnCD8β); or (vi) (pnCD8β)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8α), wherein pnCD8α is the polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnCD8β is the polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnBP is the polynucleotide encoding the binding protein, and wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or encoded self-cleaving peptides are optionally the same or different. (e.g., P2A, T2A, F2A, E2A). It should be understood that the self-cleaving peptide may comprise a linker at its N-terminus and / or C-terminus. An example of a linker is GSG. In some embodiments, a T2A peptide is provided that comprises an N-terminal GSG linker. 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.

[0239] In certain embodiments, the encoded binding protein comprises a TCR alpha chain and a TCR beta chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide positioned between a polynucleotide encoding the TCR alpha chain and a polynucleotide encoding the TCR beta chain. In further embodiments, the polynucleotide comprises: (i) (pnCD8alpha)-(pnSCP1)-(pnCD8beta)-(pnSCP2)-(pnTCRbeta)-(pnSCP3)-(pnTCRalpha); (ii) (pnCD8beta)-(pnSCP1)-(pnCD8alpha)-(pnSCP2)-(pnTCRbeta)-(pnSCP3)-(pnTCRalpha); (iii) (pnCD8alpha)-(pnSCP1)-(pnCD8beta)-(pnSCP2)-(pnTCRbeta)-(pnSCP3)-(pnTCRalpha); pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (iv)(pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pn TCRβ); (v) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β); (vi)(pnTCRβ)-(pnSCP1)-(p nTCRα)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α); (vii)(pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8α)-(pn SCP3)-(pnCD8β); (viii)(pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α), where pnCD8α is the coding polypeptide The invention further comprises a polynucleotide encoding a peptide comprising the extracellular portion of a CD8 co-receptor α chain, wherein pnCD8β is the polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor α chain, wherein pnTCRα is the polynucleotide encoding the TCRα chain, wherein pnTCRβ is the polynucleotide encoding the TCRβ chain, and wherein pnSCP1, pnSCP2, and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different. In some embodiments, SCP1 comprises SEQ ID NO.: 82, SCP2 comprises SEQ ID NO.: 74, and SCP3 comprises SEQ ID NO.: 74.

[0240] Additionally or alternatively, the polynucleotide encoding the binding protein may encode a furin cleavage site or other protease cleavage site positioned between two other polypeptides (eg, between a TCR beta chain and a TCR alpha chain).

[0241] In certain embodiments, the coded polypeptide of the present disclosure comprises one or more junction amino acids. "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, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can be produced by the design of a construct encoding a fusion protein (e.g., amino acid residues produced using restriction enzyme sites during the construction of a nucleic acid molecule encoding a fusion protein), or by cutting a self-cleaving peptide adjacent to one or more domains of an encoded binding protein of the present disclosure (e.g., a P2A peptide placed between a TCR α chain and a TCR β chain, which self-cleavage can leave one or more junction amino acids in an α chain, a TCR β chain, or both).

[0242] In another embodiment, the binding protein is expressed as part of a transgenic construct that encodes and / or the host cell of the present disclosure can encode: one or more additional auxiliary proteins, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor β chain; a CD8 co-receptor α chain or both; or any combination thereof. PCT application PCT / US2017 / 053112 describes polynucleotides and transgenic constructs that can be used to encode and express binding proteins and auxiliary components (e.g., one or more of a safety switch protein, a selection marker, a CD8 co-receptor β chain, or a CD8 co-receptor α chain), the polynucleotides, transgenic constructs, and auxiliary components (including nucleotide and amino acid sequences) of which are hereby incorporated by reference. It should be understood that any or all of the binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor β chains, or CD8 co-receptor α chains of the present disclosure can be encoded by a single nucleic acid molecule, or encoded by a polynucleotide sequence present on a separate nucleic acid molecule.

[0243] Exemplary safety switch proteins include, for example, a truncated EGF receptor polypeptide (huEGFRt), which lacks the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retains its native amino acid sequence, has a type I transmembrane cell surface localization, and has a conformationally intact binding epitope of the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011); a caspase polypeptide (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; DiStasi et al., N.E.L. Med. Am. Am.; 2011; 14:105-1110; DiStasi et al., N.E.L. Med. Am.; 2011; 14:105-1110; Wang et al., Blood 118:1255-1263, 2011); a caspase polypeptide (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005 ... .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., "Proceedings of the National Academy of Sciences of the United States of America" ​​105:623-628,2008); and marker / safety switch peptides such as RQR (CD20+CD34; Philip et al., 2014).

[0244] Other auxiliary components that can be used in the modified host cells of the present disclosure include labels or selection markers that allow identification, sorting, separation, enrichment, or tracking of cells. For example, labeled host cells with desired characteristics (e.g., antigen-specific TCRs and safety switch proteins) can be sorted from unlabeled cells in a sample and more efficiently activated and amplified for inclusion in a product of desired purity.

[0245] As used herein, the term "selection marker" comprises a nucleic acid construct (and encoded gene product) that confers a recognizable change to a cell, allowing detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker. RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR comprises a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is incorporated into the amino-terminal position (Q8) of the CD8 co-receptor stem domain. In further embodiments, the CD34 minimal binding site sequence can be combined with a target epitope of CD20 to form a dense marker / suicide gene (RQR8) for T cells (Philip et al., 2014, incorporated herein by reference). This construct allows selection of host cells expressing the construct, using, for example, CD34-specific antibodies bound to magnetic beads (Miltenyi), and utilizing the clinically accepted drug antibody rituximab, which allows selective deletion of engineered T cells expressing the transgene (Philip et al., 2014).

[0246] Additional exemplary selection markers include several truncated type I transmembrane proteins that are not normally expressed on T cells: truncated low affinity nerve growth factor, truncated CD19, and truncated CD34 (see, e.g., Di Stasi et al., NEJM 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 that they are readily available from Miltenyi Biotec's CliniMACs. TMThe availability of the selection system, which can target these markers for clinical grade sorting. However, CD19 and CD34 are relatively large surface proteins that may affect the carrier packaging capacity and transcription efficiency of the integration vector. Surface markers containing extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) can also be used. Any selection marker can be used, and should be accepted by good manufacturing practices. In certain embodiments, the selection marker is expressed with a polynucleotide encoding a gene product of interest (e.g., the binding protein of the present disclosure, such as TCR or CAR). Other examples of selection markers include, for example, reporter genes such as GFP, EGFP, β-gal or chloramphenicol acetyltransferase (CAT). In certain embodiments, selection markers, such as CD34, are expressed by cells, and CD34 can be used to select transduced cells of interest for enrichment or separation (e.g., by immunomagnetic selection), for use in methods described herein. As used herein, CD34 markers are different from anti-CD34 antibodies or, for example, scFv, TCR or another antigen recognition moiety combined with CD34.

[0247] In certain embodiments, the selection marker comprises an RQR polypeptide, truncated low affinity nerve growth factor (tNGFR), truncated CD19 (tCD19), truncated CD34 (tCD34), or any combination thereof.

[0248] With respect to RQR polypeptides, without 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 in the β chain, α chain, or both of the encoded CD8 co-receptor, or in a fragment or variant of one or both. 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 β chain containing an RQR polypeptide and further comprising a CD8 α chain.

[0249] 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 at, for example, 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 at, for example, UniProtKB-P10966 (see also UniProtKB-Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).

[0250] The isolated polynucleotides of the present disclosure may further comprise a polynucleotide encoding a safety switch protein, a selection marker, a CD8 co-receptor β chain, or a CD8 co-receptor α chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.

[0251] In any of the presently disclosed embodiments, the polynucleotides can be codon optimized for expression in a host cell. In some embodiments, the host cell comprises a human immune system cell, such as a T cell, a NK cell, or a NK-T cell (Scholten et al., Clinical Immunology 119:135, 2006). Codon optimization can be performed using known techniques and tools, for example, using OptimumGene TM Tools or GeneArt (Life Technologies, Inc.). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more codons in the codons are optimized to be expressed in a host cell) and fully codon-optimized sequences. It should be understood that in embodiments where the polynucleotide encodes more than one polypeptide (e.g., TCR α chains, TCR β chains, CD8 co-receptor α chains, CD8 co-receptor β chains, and one or more self-cleaving peptides), each polypeptide may be independently fully codon-optimized, partially codon-optimized, or not codon-optimized.

[0252] The amino acid and polynucleotide sequences of exemplary binding proteins "11N4A" and "11N6" are shown in Table 1.

[0253] Table 1: Certain polynucleotide and amino acid sequences associated with TCRs 11N4A and 11N6

[0254]

[0255]

[0256] 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 may be heterologous to the sequence of (ii). The sequence of (ii) may be codon-optimized, for example, for expression in human T cells.

[0257] carrier

[0258] In another aspect, the present disclosure provides an expression vector comprising any polynucleotide as provided herein operably linked to an expression control sequence.

[0259] Also provided herein are carriers comprising polynucleotides of the present disclosure or transgenic constructs. Some examples of carriers include plasmids, viral vectors, clays, etc. Some carriers may be able to autonomously replicate (for example, bacterial vectors and episomal mammalian vectors with bacterial replication origin) in the host cell into which they are introduced, and other carriers may be integrated into the genome of the host cell, or promote the integration of polynucleotide inserts when introduced into the host cell, and thereby replicate together with the host genome (for example, slow viral vectors, retroviral vectors). In addition, some carriers can instruct the expression (these carriers may be referred to as "expression vectors") of the gene operably connected thereto. According to relevant embodiments, it should be further understood that if one or more medicaments (for example, polynucleotides encoding polypeptides as described herein) are administered to a subject jointly, each medicament may be present in a separate or identical carrier, and multiple carriers (each carrier containing different medicaments or identical medicaments) may be introduced into a cell or cell colony or administered to a subject.

[0260] In certain embodiments, the polynucleotides of the present disclosure may be operably linked to certain elements of a vector. For example, the polynucleotide sequences required to achieve expression and processing of the coding sequence to which the polynucleotide sequence is linked may be operably linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective 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 possible sequences that enhance protein secretion. An expression control sequence may be operably linked if it is adjacent to a gene of interest and an expression control sequence that acts in trans or at a distance to control the gene of interest.

[0261] 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 and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses); positive-strand RNA viruses such as picornaviruses and alphaviruses; and double-stranded DNA viruses including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., cowpox, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepadnaviruses. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type viruses, B-type viruses, D-type viruses, HTLV-BLV groups, lentiviruses, and foamy viruses (Coffin, JM, Retroviridae: The viruses and their replication, in Fundamental Virology, 3rd ed.; BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0262] "Retrovirus" is a virus with an RNA genome that is reverse-transcribed into DNA using a reverse transcriptase, and the reverse-transcribed DNA is subsequently incorporated into the host cell genome. "γ-retrovirus" refers to a genus of the Retroviridae family. Examples of γ-retroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendothelioma virus. As used herein, "slow virus vector" means a slow virus vector based on HIV for gene delivery, which can be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a range of different cell types. Slow virus vectors are typically produced after three (packaging, envelope, and transfer) or more plasmids are transiently transfected into production cells. Similar to HIV, slow virus vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, viral RNA undergoes reverse transcription, which is mediated by a viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the matrix into which the virus integrates into the DNA of infected cells. In some embodiments, the lentiviral vector is a self-inactivating lentiviral vector. The self-inactivating lentiviral vector can include a modification to prevent the enhancer and promoter elements in the 5' long terminal repeat (LTR) of the vector from being transferred to the transduced cells, for example, after one round of reverse transcription, the deletion in the 3'LTR of the viral genome is transferred to the 5'LTR, producing a provirus that does not contain LTR-derived enhancer or promoter elements. In some embodiments, the lentiviral vector is a third generation lentiviral vector. The third generation lentiviral vector can utilize a packaging system that is divided into two or more plasmids, for example, one encoding Rev and one encoding Gag and Pol. The third generation lentiviral vector can utilize a packaging system that lacks Tat or does not require Tat expression, but instead comprises a chimeric 5'LTR fused to a heterologous promoter on a transfer plasmid, for example.

[0263] In certain embodiments, the viral vector can be a gammaretrovirus, such as a Moloney murine leukemia virus (MLV)-derived vector. In other embodiments, the viral vector can 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 of using retroviral and lentiviral 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 the following literature: U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., Journal of Immunology 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Molecular Therapy 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. Other viral vectors can also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0264] Other vectors developed for gene therapy applications can also be used with the compositions and methods of the present disclosure. Such vectors include vectors derived from baculovirus and alphavirus. (Jolly, D. J. 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).

[0265] When the viral vector genome comprises multiple polynucleotides to be expressed as independent transcripts in the host cell, the viral vector may also comprise additional sequences between the two (or more) transcripts, thereby allowing bicistronic or polycistronic expression. Examples of such sequences for use in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0266] In certain embodiments, the vector is capable of delivering a polynucleotide or transgenic construct to a host cell (e.g., a hematopoietic progenitor cell or a human immune system cell). In specific embodiments, the vector is capable of delivering a polynucleotide or transgenic construct to a human immune system cell, such as a CD4 + T cells, CD8 + T cells, CD4 - CD8 - In some embodiments, the present invention provides a method for the treatment of a double negative T cell, a stem cell memory T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof. In a further embodiment, the vector can deliver the transgenic construct to naive T cells, central memory T cells, effector memory T cells, or any combination thereof. In certain embodiments, the vector encoding the polynucleotides of the present disclosure or the transgenic construct may further include a polynucleotide encoding a nuclease, which may be used to carry out chromosome knockout (e.g., CRISPR-Cas endonucleases or another endonucleases as disclosed herein) in a host cell, or may be used to deliver therapeutic polynucleotides or transgenics or its portion to a host cell in gene therapy replacement or gene repair therapy. Alternatively, the nuclease for chromosome knockout or gene replacement or gene repair therapy may be delivered to a host cell independently of the vector encoding the polynucleotides of the present disclosure or the transgenic construct.

[0267] In certain embodiments, the vector is capable of delivering the polynucleotide to a host cell. In further embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In still further embodiments, the human immune system cell is a CD4+T cell, a CD8+T cell, a CD4-CD8-double negative T cell, a γδT cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof. In yet further embodiments, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0268] In any of the presently disclosed embodiments, the vector is a viral vector. In certain embodiments, the viral vector is a lentiviral vector or a gamma-retroviral vector.

[0269] Examples of transposon-based systems that can be used include, but are not limited to, Sleeping Beauty (e.g., derived from the genome of salmon); Piggyback (e.g., derived from Lepidoptera cells and / or the little brown bat (Myotis lucifugus)); Mariner (e.g., derived from Drosophila); Frog Prince (e.g., derived from Rana pipiens); Tol2 (e.g., derived from medaka fish); and spinON.

[0270] host cells

[0271] Also provided herein are host cells encoding and / or expressing binding proteins (and optionally one or more accessory proteins, such as transduction markers, CD8 co-receptor polypeptides, etc., as provided herein). In certain embodiments, a host cell is provided that is modified to contain a polynucleotide and / or expression vector of the present disclosure, and / or to express a binding protein of the present disclosure.

[0272] Any suitable host cell can be modified to include a heterologous polynucleotide encoding a binding protein of the present disclosure, including, for example, an immune cell, such as a T cell, NK cell, or NK-T cell, modified to include a heterologous polynucleotide. In some embodiments, the modified immune cell comprises a CD4 + T cells, CD8 + T cells or both. Methods for transfecting / transducing T cells with a desired nucleic acid have been described (e.g., U.S. Patent Application Publication No. US2004 / 0087025) as adoptive transfer procedures using T cells with the 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. Gen. Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US2011 / 0243972; US 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), such that based on the teachings herein, it is contemplated that these methods may be adapted to the presently disclosed embodiments.

[0273] Any suitable method can be used to transfect or transduce cells, such as T cells, or to administer the polynucleotides or compositions of the present method. Known methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid molecules, and certain commercial products, such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, ultrasound loading, liposome-mediated transfection, receptor-mediated transduction, microparticle bombardment, transposon-mediated transfer, and the like. Still other methods for transfecting or transducing host cells employ vectors, which will be described in further detail herein.

[0274] In certain embodiments, the host cell or modified cell comprises hematopoietic progenitor cells, stem cells (e.g., iPSC) and / or human immune cells. In certain embodiments, immune cells include T cells, NK cells, NK-T cells, dendritic cells, macrophages, monocytes, or any combination thereof. In further embodiments, immune cells include CD4+T cells, CD8+T cells, CD4-CD8- double negative T cells, γδT cells, or any combination thereof. In certain further embodiments, immune cells include CD4+T cells and CD8+T cells. In certain still further embodiments, CD4+T cells, CD8+T cells, or both include (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or includes a CD8 co-receptor α chain; (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or includes a CD8 co-receptor β chain; or (iii) the polynucleotides of (i) and the polynucleotides of (ii).

[0275] The host cell may be a peripheral blood mononuclear cell (PBMC). The host cell may be a lymphoid cell. The host cell may be a lymphocyte. The host cell may be a T cell. The host cell may be an αβ T cell (whether or not expressing an endogenous α-β TCR). The host cell may be a γδ T cell (whether or not expressing an endogenous γ-δ TCR). The host cell may be a B cell. The host cell may be a natural killer (NK) cell. The host cell may be a natural killer T (NKT) cell. The host cell may be a mammalian cell. The host cell may be a human cell.

[0276] 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, or a hematopoietic stem cell (HSC).

[0277] In any of the above embodiments, the host cell (e.g., immune cell) can be modified to reduce or eliminate the expression of one or more endogenous genes encoding polypeptides involved in immune signaling or other related activities. Exemplary gene knockouts include gene knockouts encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA molecules, TCR molecules, etc. Without wishing to be bound by theory, some endogenously expressed immune cell proteins can be recognized as foreign by the allogeneic host receiving the modified immune cells, which may result in the 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 the heterologously expressed binding proteins of the present disclosure (e.g., endogenous TCR of modified T cells, binding to non-Ras antigens, and thereby interfering with the modified immune cells bound to cells expressing Ras antigens).

[0278] 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 an autologous or allogeneic host environment and can 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 knockout of one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, an HLA component (e.g., a gene encoding α1 macroglobulin, α2 macroglobulin, α3 macroglobulin, β1 microglobulin, or β2 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 therapies of which are incorporated herein by reference in their entireties).

[0279] As used herein, the term "chromosomal gene knockout" refers to a genetic alteration in a host cell or an introduced inhibitor that prevents (e.g., reduces, delays, inhibits, or eliminates) the host cell from producing a functionally active endogenous polypeptide product. The alteration that results in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and chain breaks, as well as heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0280] In certain embodiments, chromosome gene knockout or gene knock-in is performed by chromosome editing of the host cell. Chromosome editing can be performed using, for example, endonucleases. As used herein, "endonuclease" refers to an enzyme capable of catalyzing the cutting of phosphodiester bonds within a polynucleotide chain. In certain embodiments, endonucleases can cut the targeted gene, thereby inactivating or "knocking out" the targeted gene. Endonucleases can be naturally occurring, recombinant, genetically modified, or fusion endonucleases. The nucleic acid chain breaks caused by endonucleases are typically repaired by different mechanisms of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, donor nucleic acid molecules can be used for donor gene "knocking in", for target gene "knocking out", and optionally inactivating the target gene by donor gene knocking in or target gene knockout events. NHEJ is a kind of error-prone repair process, which usually results in changes in the DNA sequence at the cleavage site, such as substitution, deletion, or addition of at least one nucleotide. NHEJ can be used for "knocking out" the target gene. Examples of endonucleases include zinc finger nucleases, TALE nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0281] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein comprising a zinc finger DNA binding domain fused to a non-specific DNA cleavage domain, such as Fok1 endonuclease. Each zinc finger motif of approximately 30 amino acids binds to approximately 3 base pairs of DNA, and the amino acids at certain residues can be varied to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proceedings of the National Academy of Sciences of the United States of America 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285:1917-1934, 1999). Multiple zinc finger motifs can be connected in series to generate binding specificity for a desired DNA sequence, such as regions ranging in length from about 9 to about 18 base pairs. As background, ZFN mediates genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome and promotes the targeted integration of transgenes containing flanking sequences homologous to the genome at the site of the DSB by homology-directed repair. Alternatively, the DSBs generated by ZFNs can be repaired by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, gene knockout comprises insertions, deletions, mutations, or combinations thereof performed using ZFN molecules.

[0282] 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 FokI endonuclease. " TALE DNA binding domain " or " TALE " is composed of one or more TALE repeat domains / units, each of which typically has a highly conserved 33-35 amino acid sequence, in which the 12th and 13th amino acids are different. The TALE repeat domain participates in the binding of TALE to the target DNA sequence. Different amino acid residues, referred to as repeat variable diresidues (RVDs), are associated with specific nucleotide recognition. The natural (canonical) code for the DNA recognition of these TALEs has been determined so that the HD (histidine-aspartic acid) sequence at position 12 and position 13 of TALE causes TALE to bind to cytosine (C), NG (asparagine-glycine) to bind to T nucleotides, NI (asparagine-isoleucine) to bind to A, NN (asparagine-asparagine) to bind to G or A nucleotides, and NG (asparagine-glycine) to bind to T nucleotides. Non-canonical (atypical) RVDs are also known (see, for example, U.S. Patent Publication No. US2011 / 0301073, which is incorporated herein by reference in its entirety). TALEN can be used to direct site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) connects DNA from both sides of the double-strand break, with little or no sequence overlap for annealing, thereby introducing errors in knocking out gene expression. Alternatively, homology-directed repair can introduce a transgene at the site of the DSB, provided that there are homologous flanking sequences in the transgene. In certain embodiments, gene knockout comprises insertions, deletions, mutations, or a combination thereof, and is performed using TALEN molecules.

[0283] As used herein, "Clustered Regularly Interspaced Short Palindromic Repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that uses CRISPR RNA (crRNA)-guided Cas nucleases to recognize target sites (called protospacers) within the genome through base pairing complementarity, and then cuts the DNA if a short, conserved protospacer-associated motif (PAM) is immediately following the 3' of the complementary target sequence. The CRISPR / Cas system is divided into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nuclease. The crRNA-guided monitoring complex in types I and III requires multiple Cas subunits. The most studied type II system comprises at least three components: an RNA-guided Cas9 nuclease, crRNA, and a trans-acting crRNA (tracrRNA). tracrRNA comprises a duplex-forming region. CrRNA and tracrRNA form a duplex, which can interact with Cas9 nuclease and guide Cas9 / crRNA:tracrRNA complex to a specific site on target DNA by Watson-Crick base pairing (Watson-Crick base-pairing) between the spacer on crRNA upstream of PAM and the original spacer on target DNA. Cas9 nuclease cuts the double-stranded breaks in the region defined by the crRNA spacer. The repair of NHEJ causes insertion and / or deletion, which destroys the expression of the targeted gene seat. Alternatively, a transgene with a homologous flanking sequence can be introduced at the site of DSB by homology-directed repair. CrRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, for example, Jinek et al., Science 337:816-21, 2012). In addition, the region of the guide RNA that is complementary to the target site can be altered or programmed to target the desired sequence (Xie et al., PLoS One 9:e100448, 2014; U.S. Patent Application Publication No. US2014 / 0068797, U.S. Patent Application Publication No. US2014 / 0186843; U.S. Patent No. 8,697,359 and PCT Publication No. WO 2015 / 071474; each of which is incorporated by reference). In certain embodiments, gene knockout comprises an insertion, deletion, mutation, or a combination thereof and is performed using a CRISPR / Cas nuclease system.

[0284] Exemplary gRNA sequences and methods of using them to knock out endogenous genes encoding immune cell proteins include the exemplary gRNA sequences and methods described in Ren et al., Clin Cancer Res 23(9): 2255-2266 (2017), the gRNAs, CAS9 DNA, vectors, and gene knockout technologies of which are hereby incorporated by reference in their entirety.

[0285] As used herein, "meganucleases," also known as "homing endonucleases," refer to endodeoxyribonucleases characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Based on sequence and structural motifs, nucleases can be divided into five families: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-Scel, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-ScelI, I-PpoI, I-ScelIII, I-Crel, I-TevI, I-TevII, and I-TevIII, whose recognition sequences are known (see, 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 in Genetics). 1996; Gimble et al., Journal of Mol. Biol. 263:163-180, 1996; Argast et al., Journal of Mol. Biol. 280:345-353, 1998).

[0286] In certain embodiments, naturally occurring meganucleases can be used to promote site-specific genomic modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, a gene encoding HLA, or a gene encoding a TCR component. In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genomic modification (see, e.g., Porteus et al., Nature Biotechnology 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., Mol. Cell 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Therapy 442:656-659, 2006). Ther.) 7:49-66, 2007; U.S. Patent Publication Nos. US2007 / 0117128; US2006 / 0206949; US2006 / 0153826; US2006 / 0078552; and US 2004 / 0002092). In another embodiment, chromosomal gene knockouts are generated using homing endonucleases that have been modified with the modular DNA binding domains of TALENs to prepare fusion proteins termed 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 conjunction with an exogenous donor template encoding a polypeptide of interest.

[0287] In certain embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell), wherein the host cell 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 wherein 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.

[0288] In certain embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is performed 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 method that uses components from the CRISPR system together with other enzymes to directly introduce point mutations into cellular DNA or RNA without breaking double-stranded DNA. Certain DNA base editors comprise a catalytically inactive nuclease fused to a nucleobase deaminase and, in some cases, a DNA glycosidase inhibitor. RNA base editors function similarly, using components that target RNA. Base editors directly convert one base or base pair to another, enabling efficient installation of point mutations in non-dividing cells without producing excess, undesirable editing byproducts. See, e.g., Rees H et al. Nature Reviews Genetics (2018).

[0289] Chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells after the use of a knockout procedure or agent. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., absence of mRNA or polypeptide products encoded by the gene) after knockout.

[0290] In certain embodiments, the chromosomal gene knockout comprises a knockout of an HLA component gene selected from the group consisting of an α1 macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a β1 microglobulin gene, or a β2 microglobulin gene.

[0291] In certain embodiments, the chromosomal gene knockout comprises a knockout of a TCR component gene selected from the group consisting of a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or a combination thereof.

[0292] 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 12-fold, at least 13-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 21-fold, at least 22-fold, at least 23-fold, at least 24-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 least 43-fold The host cell can comprise a binding protein (e.g., a TCR comprising Vα 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, for example, present in a peptide:HLA complex). The increase in avidity can be, for example, as determined by an assay for determining expression of an activation marker (e.g., CD137, CD69, granzyme B, CD107a, IFN-γ, TNF-α, IL-12, cytokines, interleukins, interferons) upon exposure to a target cell expressing or presenting the target antigen, and / or an assay for determining EC50 (e.g., the dose of peptide that achieves half-maximal activation of a T cell population). In some embodiments, both the host cell and the control cell are T cells, and the host cell population and the control cell population can comprise the same, about the same, or substantially the same composition or number of T cell types (e.g., CD4+, CD8+, or both).

[0293] 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 target cell killing compared to a population of control cells (e.g., cells expressing a control binding protein that is specific for the same target antigen). For example, killing of target cells can be, e.g., as determined by an in vitro cytotoxicity assay, e.g., at an effector to target ratio of 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, both the host cells and the control cells are T cells, and the host cell population and the control cell population can comprise the same, about the same, or substantially the same composition or number of T cell types (e.g., CD4+, CD8+, or both).

[0294] 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 12-fold, at least 13-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 21-fold, at least 22-fold, at least 23-fold, at least 24-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 least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 51-fold, at least 52-fold, at least 53-fold, at least 54-fold, at least 55-fold, at least 56-fold, at least 57-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold or at least 5000-fold increased activation. Activation can be, for example, as determined by determining that an activation marker (e.g., CD137, CD69, granzyme B, CD107a, IFN-γ, TNF-α, IL-12, cytokine, interleukin, interferon) is expressed when exposed to a target cell expressing or presenting a target antigen. In some embodiments, both the host cells and the control cells are T cells, and the host cell population and the control cell population can comprise the same, about the same, or substantially the same composition or number of T cell types (e.g., CD4+, CD8+, or both).

[0295] In some embodiments, a population of host cells comprising a binding protein disclosed herein is resistant to exhaustion, e.g., exhibits effective tumor cell killing upon multiple rechallenges 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 rechallenges), or exhibits sustained control of tumor growth in vivo.

[0296] In some embodiments, a population of host cells comprising a binding protein disclosed herein is resistant to exhaustion, e.g., exhibits superior tumor cell killing upon multiple rechallenges 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 rechallenges), or exhibits superior control of tumor growth in vivo, compared to a population of control cells. In some embodiments, both the host cells and the control cells are T cells, and the host cell population and the control cell population can comprise the same, about the same, or substantially the same composition or number of T cell types (e.g., CD4+, CD8+, or both).

[0297] Host cell compositions and unit dosages

[0298] 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.

[0299] 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 + T cell compositions 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% of modified CD8 + The compositions of T cells are combined in a ratio of about 1:1, wherein the unit dose contains a reduced amount of or is substantially free of naive T cells (i.e., the population of naive T cells present in the unit dose is 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% compared to a patient sample having a comparable number of PBMCs).

[0300] In some embodiments, the host cell composition or unit dose comprises (i) at least about 50% modified CD4 + T cell composition comprising at least about 50% modified CD8 + In another embodiment, the host cell composition or unit dose comprises (i) at least about 60% modified CD4 T cells. + T cell composition comprising at least about 60% modified CD8 + In yet other embodiments, the host cell composition or unit dose comprises (i) at least about 70% engineered CD4 T cells. + A composition of T cells comprising 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. + T cell composition comprising at least about 80% modified CD8+ In some embodiments, the host cell composition or unit dose comprises (i) at least about 85% modified CD4 T cells. + T cell composition comprising at least about 85% modified CD8 + In some embodiments, the host cell composition or unit dose comprises (i) at least about 90% modified CD4 T cells. + T cell composition comprising at least about 90% modified CD8 + The host cell compositions are combined in a ratio of about 1:1, wherein the host cell composition or unit dose contains a reduced amount of or is substantially free of naive T cells.

[0301] In some embodiments, the composition comprises a CD4+ cell population comprising (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+ T cells. In some embodiments, the composition further comprises a CD8+ cell population comprising (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.

[0302] In some embodiments, the host cell composition or unit dose comprises a CD4+ to CD8+ T cell ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 3:2, or about 2:3 (e.g., CD4+ T cells modified to comprise or express a binding protein disclosed herein and CD8+ T cells modified to comprise or express a binding protein disclosed herein).

[0303] In some embodiments, the host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells of 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.

[0304] In some embodiments, the host cell composition or unit dose comprises a ratio of CD4+ to CD8+ T cells of 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.

[0305] In some embodiments, the host cell composition or unit dose comprises CD4+ to CD8+ T cells at a ratio of 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 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 10 and 1:5, 1:10 and 1:7, 1:5 and 10:1, 1:5 and 8:1, 1:5 and 7:1, 1:5 and 6:1, 1:5 and 5:1, 1:5 and 4:1, 1:5 and 3:1, 1:5 and 2:1, 1:5 and 1:1, 1:5 and 1:2, 1:5 and 1:3, 1:5 and 1:4, 1:3 and 10:1, 1:3 and 8:1, 1:3 and 7:1, 1:3 and 6:1, 1:3 and 5:1, 1:3 and 4:1, 1:3 and 3:1, 1:3 and 2:1, 1:3 and 1:1, 1:3 and 1:2, 1:2 and 10:1, 1:2 and 8:1, 1:2 and 7:1, 1:2 and 6:1, 1:2 and 5:1, 1:2 and 4:1, 1:2 and 3:1, 1:2 and 2:1, 1:2 and 1:1, 1:1 and 10:1, 1:1 and 8:1, 1:1 and 7:1, 1:1 and 6:1, 1:1 and 5:1, 1:1 and 4: 1, 1:1 and 3:1, 1:1 and 2:1, 2:1 and 10:1, 2:1 and 8:1, 2:1 and 7:1, 2:1 and 6:1, 2:1 and 5:1, 2:1 and 4:1, 2:1 and 3:1, 3:1 and 10:1, 3:1 and 8:1, 3:1 and 7:1, 3:1 and 6:1, 3:1 and 5:1, 3:1 and 4:1, 5:1 and 10:1, 5:1 and 8:1, 5:1 and 7:1, or 5:1 and 6:1.

[0306] The CD4+ T cells in the composition, host cell composition, or unit dose can be CD4+ T cells modified or engineered, for example, using the vectors or polynucleotides disclosed herein, to express a CD8 co-receptor disclosed herein.

[0307] It should be understood that the host cell compositions or unit doses of the present disclosure can comprise any host cell described herein, or any combination of host cells. In certain embodiments, for example, the host cell compositions or unit doses comprise modified CD8+ T cells, modified CD4+ T cells, or both, wherein the T cells are modified to encode a peptide specific for Ras:HLA-A. *11:01 complex has a specific binding protein. Additionally or alternatively, the host cell composition or unit dose of the present disclosure can comprise any host cell or combination of host cells as described herein, and can further comprise modified cells (e.g., immune cells, such as T cells) that express antibodies against different antigens (e.g., different Ras antigens, or antigens from different proteins or targets, such as 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, LewisA, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including 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, tyrosine kinase inhibitors bound to HLA Acidase peptide, WT-1 peptide 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 1 -H, Robol, α-fetoprotein (AFP), Frizzled, OX40, PRAME and SSX-2, etc. For example, a unit dose may contain a modified CD8 + T cells and modified CD4 T cells expressing a binding protein (e.g., CAR) that specifically binds to the PSMA antigen + T cells (and / or modified CD8 + T cells). It is also understood that any of the host cells disclosed herein can be administered in combination therapy.

[0308] In any of the embodiments described herein, the host cell composition or unit dose comprises an equal or approximately equal number of engineered CD45RA- CD3 + CD8 + and modified CD45RA - CD3 + CD4 + T M cell.

[0309] In any of the embodiments described herein, the host cell composition or unit dose comprises one or more populations of cells selected for CD62L positiveness (eg, CD4+ or CD8+ cells), eg, to enhance in vivo persistence.

[0310] The host cell can be genetically engineered to contain or express the binding protein in vitro, in vitro, or in vivo. In some embodiments, the host cell is genetically engineered to express the binding protein in vitro. In some embodiments, the host cell is genetically engineered to express the binding protein in vitro. In some embodiments, the host cell is genetically engineered to express the binding protein in vivo.

[0311] use

[0312] In another aspect, the present disclosure provides a method for treating or preventing recurrence of a disease or condition associated with a KRAS G12V or NRAS G12V mutation or a HRAS G12V mutation in a subject. Such diseases or conditions include, for example, cancer, such as solid cancers and hematological malignancies. In certain exemplary embodiments, the disease or condition comprises pancreatic cancer (or pancreas carcinoma), optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer (or colorectal carcinoma); colon cancer; colorectal adenocarcinoma; lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer (or endometrial 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 intestinal cancer; breast cancer (or breast carcinoma); melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid carcinoma; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer carcinoma); 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 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; thyroid malignant neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma or papillary thyroid carcinoma. Diseases or conditions that can be treated by the compositions or methods disclosed herein include advanced or metastatic forms of the cancers disclosed herein.

[0313] "Treat" or "treatment" or "ameliorate" 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). In general, an appropriate dose or treatment regimen comprising the compositions of the present disclosure (e.g., comprising binding proteins, polynucleotides, vectors, host cells, host cell compositions, unit doses and / or immunogenic polypeptides) is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefits include improved clinical outcome; alleviation or alleviation of symptoms associated with the disease; reduction in the occurrence of symptoms; improved quality of life; a longer disease-free state; a reduction in the extent of the disease, stabilization of the disease state; slowing of disease progression; remission; survival; prolonged survival; or any combination thereof.

[0314] As used herein, "therapeutically effective amount" or "effective amount" refers to an amount of a composition sufficient to produce a therapeutic effect, including: improved clinical outcome; alleviation or relief of symptoms associated with the disease; reduction in the occurrence of symptoms; improved quality of life; longer disease-free state; reduction in the extent of the disease; stabilization of the disease state; slowing of disease progression; remission; survival; or statistically significant extension of survival. When referring to a single active ingredient or a cell expressing a single active ingredient administered alone, the therapeutically effective amount refers to the effect of the ingredient or the cell expressing the ingredient alone. When referring to a combination, the therapeutically effective amount refers to the combined amount of the active ingredient or the auxiliary active ingredient of the combination and the cell expressing the active ingredient that produces the therapeutic effect, whether administered sequentially or simultaneously. The combination can also be a cell expressing more than one active ingredient.

[0315] The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refers to a biocompatible vehicle, such as physiological saline, which is described in more detail herein, that is suitable for administration to humans or other non-human mammalian subjects and is generally recognized as safe or not to cause serious adverse events.

[0316] As used herein, "statistically significant" means a p-value of 0.050 or less when calculated using a Students t-test or other appropriate statistical test, and indicates that the particular event or result measured is unlikely to have occurred by chance.

[0317] In general, the subjects that can be treated by the present invention are 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 can be at any suitable age, including infants, teenagers, adolescents, adults and elderly subjects. The subject can be a mammal. As determined by those skilled in the art of medicine, the pharmaceutical composition according to the present disclosure can be administered in a manner suitable for the disease to be treated, the condition or illness. In any of the foregoing embodiments, the modified host cells, host cell compositions or unit doses as described herein are administered intravenously, intraperitoneally, intratumorally, intramedullary, intralymph nodeally or intracerebrospinal 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 illness; the specific form of the active ingredient; and the method of administration.

[0318] As used herein, the term "adoptive immune therapy" or "adoptive immunotherapy" refers 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 (immune cells are from the recipient), allogeneic (immune cells are from a donor of the same species but different from the recipient), or syngeneic (immune cells are from a donor that is genetically identical or substantially genetically identical to the recipient, such as monozygotic twins).

[0319] 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.

[0320] In some embodiments, the subject is HLA-A * 11 + (e.g., HLA-A * 11:01 + ).

[0321] In certain embodiments, the method comprises determining the presence of one or more HLA types and / or identifying Ras antigens in the subject prior to administering therapy according to the present disclosure. In some embodiments, for example, prior to administering therapy, the presence of one or more HLA types and / or Ras antigens (e.g., G12V mutations) in the subject has been determined, and therapy is administered based at least in part on the presence of the HLA type and / or Ras antigens. In some embodiments, the method further comprises genotyping the subject's tumor for the KRAS G12 allele prior to administration. In some cases, it is determined prior to administration that the subject carries the KRAS G12V allele.

[0322] The expression of HLA alleles can be determined by, for example, gene 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 by molecular methods in clinical laboratories that have obtained HLA typing licenses. In some embodiments, HLA typing is performed using PCR amplification, followed by high-throughput NGS and subsequent HLA determination. Here, HLA haplotypes can be determined at the major HLA loci (e.g., HLA-A, HLA-B, HLA-C, etc.).

[0323] HLA typing can be performed using any known method, including, for example, protein or nucleic acid detection. Examples of nucleic acid detection 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, methods disclosed in Mayor et al., PLOS ONE, 10(5):e0127153 (2015), the methods and reagents of which are incorporated herein by reference.

[0324] In certain embodiments, the method comprises when the subject expresses HLA-A * At 11:01, a composition comprising modified CD8+ and / or modified CD4+ T cells comprising a heterologous polynucleotide encoding a second binding protein as provided herein is administered.

[0325] 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 subsets (e.g., optionally comprising memory and / or naive CD8 + T cells); a modified CD4 + T cell subsets (e.g., optionally comprising memory and / or naive CD4 + T cells) or more typically greater than 10 2 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 more than 10 10 In certain embodiments, about 10 4 to about 10 10 cells / m 2 range, preferably about 10 5 to about 10 9 cells / m 2 In some embodiments, the administered dose comprises up to about 3.3 x 10 5 In some embodiments, the administered dose comprises up to about 1 x 10 6 cells / kg. In some embodiments, the administered dose comprises up to about 3.3 x 10 6 In some embodiments, the administered dose comprises up to about 1 x 10 7 In certain embodiments, the cell-free 4 cells / kg, 5x10 5 cells / kg, 5x10 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 of at least about 5 x 10 cells / kg. 4 cells / kg, 5x10 5 cells / kg, 5x10 6 cells / kg or up to approximately 5 x 10 7The modified immune cells are administered to a subject at a dose of 100 cells / kg. The number of cells will depend on the desired end use of the composition, as well as the type of cells included therein. For example, the cells modified to contain the binding protein can comprise 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 such cells. For the uses provided herein, the volume of the cells is typically one liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In some embodiments, the density of the desired cells is typically greater than 10 4 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 cells / ml or greater. The cells can be administered as a single infusion or in multiple infusions over a period of time. Clinically relevant numbers of immune cells can be distributed to cumulatively equal to or greater than 10 6 10 7 10 8 10 9 10 10 or 10 11 In certain embodiments, the unit dose of modified immune cells can be co-administered with hematopoietic stem cells from an allogeneic donor (e.g., simultaneously or concurrently). In some embodiments, one or more of the modified immune cells contained in the unit dose are autologous to the subject.

[0326] In some embodiments, a unit dose comprises, consists essentially of, or consists of, or multiple unit doses comprise, consist essentially of, or consist of: 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 living host cells, e.g., encoding, comprising, or expressing a binding protein as disclosed herein.

[0327] In some embodiments, a unit dose comprises, consists essentially of, or consists of, or multiple unit doses comprise, consist essentially of, or consist of: 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 living host cells, e.g., encoding, comprising, or expressing a binding protein as disclosed herein.

[0328] In some embodiments, a unit dose comprises, consists essentially of, or consists of, or a plurality of unit doses comprises, consists essentially of, or consists of: about 1 x 10^8, about 5 x 10^8, about 1 x 10^9, about 2 x 10^9, about 3 x 10^9, about 4 x 10^9, about 5 x 10^9, about 6 x 10^9, about 7 x 10^9, about 8 x 10^9, about 9 x 10^9, about 1 x 10^10, about 1.1 x 10^10, about 1.2 x 10^10 ^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 living host cells, e.g., encoding, comprising or expressing a binding protein as disclosed herein.

[0329] In some embodiments, a unit dose comprises, consists essentially of, or consists of, or a plurality of unit doses comprises, consists essentially of, or consists of: 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 about 5x10^9, about 5x10^9 to about 1x10^11, about 5x10^9 to about 5x10^10, about 5x10^9 to about 2x10^10, about 5x10^9 to about 1.5x10^10, about 5x10^9 to about 1x10^10, about 1x10^10 to about 1x10^11, about 1x10^10 to about 5x10^10, about 1x10^10 to about 2x10^10, about 1x10^10 to about 1.5x10^10, about 1.5x10^10 to about 1x10^11, about 1.5x10^10 to about 5x10^10, or about 1.5x10^10 to about 2x10^10, for example, living host cells encoding, comprising or expressing a binding protein as disclosed herein.

[0330] In some embodiments, the subject receiving the modified immune cells has previously received lymphodepleting chemotherapy. In further embodiments, the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0331] In some embodiments, the method further comprises administering to the subject an inhibitor of an immune checkpoint molecule as disclosed herein.

[0332] Also contemplated are pharmaceutical compositions (i.e., compositions) comprising a composition as disclosed herein (binding protein, polynucleotide, vector, host cell, host cell composition, unit dose, and / or immunogenic polypeptide) and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextrose, glycerol, and the like, and combinations thereof. In an embodiment, 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), and 5% dextrose in water, Ringer's lactate, can be used. The infusion medium can be supplemented with human serum albumin or other human serum components.

[0333] As determined by those skilled in the art of medicine, pharmaceutical compositions can be administered in a manner suitable for the disease or condition to be treated (or prevented). Suitable dosages of the compositions and 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 specific form of the active ingredient, and the method of administration. Typically, suitable dosages and treatment regimens provide compositions in an amount sufficient to provide therapeutic and / or preventive benefits (as described herein, including improved clinical outcomes, such as complete or partial remissions that are more frequent, or disease-free and / or overall survival is longer, or symptom severity is alleviated).

[0334] As described herein, an effective amount of a pharmaceutical composition refers to an amount sufficient to achieve the desired clinical outcome or beneficial treatment at the desired dosage and within the desired time period. An effective amount can be delivered in one or more administrations. If administration is for a subject known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used to refer to treatment, while a "prophylactic effective amount" can be used to describe an effective amount applied as a preventive process to a subject susceptible to or at risk of a disease or disease state (e.g., recurrence).

[0335] The pharmaceutical compositions described herein can be presented in unit dose or multi-dose containers, such as sealed ampoules or vials. Such containers can be refrigerated to maintain the stability of the formulation until infusion into the patient. Dosages will vary, but a preferred dose for administering the modified immune cells described herein is about 10 4 cells / m 2 , about 5x10 4 cells / m 2 , about 10 5 cells / 2 , about 5x10 5 cells / 2 , about 10 6 cells / 2 , about 5x10 6 cells / m 2 , about 10 7 cells / m 2 , about 5x10 7 cells / m 2 , about 10 8 cells / m 2 , about 5x10 8 cells / m 2 , about 10 9 cells / m 2 , about 5x10 9 cells / m 2 , about 10 10 cells / m 2 , about 5x10 10 cells / m 2 or about 10 11 cells / m 2 In certain embodiments, a unit dose comprises about 10 4 cells / m 2 to about 10 11 cells / m 2 Develop appropriate dosing and treatment regimens for using the specific compositions described herein in various treatment regimens, including, for example, parenteral or intravenous administration or formulation.

[0336] If the subject composition is administered parenterally, the composition may also include a sterile aqueous solution or an oily solution or suspension. Suitable non-toxic parenteral acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol or a mixture of polyethylene glycol and water. The aqueous solution or suspension may further comprise one or more buffers, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in the preparation of any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in terms of the amount used. In addition, the active compound can be incorporated into sustained-release formulations and formulations. As used herein, dosage unit form refers to a physically separate unit suitable as a single dose (unitary dosage) of a subject to be treated; each unit can contain a predetermined amount of engineered immune cells or active compounds (which are calculated to produce the desired effect in combination with an appropriate pharmaceutical carrier).

[0337] In general, appropriate dosages and treatment regimens provide active molecules or cells in amounts sufficient to provide benefit. Such responses 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 pre-existing immune responses to tumor proteins is generally associated with improved clinical outcomes. Such immune responses can generally be assessed using conventional standard proliferation, cytotoxicity, or cytokine assays.

[0338] For prophylactic use, the dosage should be sufficient to prevent the disease associated with the disease or condition, delay its onset, or lessen its severity. The prophylactic benefit of the immunogenic compositions administered according to 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 by appropriate statistical, biological, and clinical methods and techniques, all of which can be readily practiced by those skilled in the art.

[0339] As used herein, administration of a composition refers to delivering it to a subject, regardless of the route or mode of delivery. Administration can be performed continuously or intermittently, as well as parenterally. The composition can be administered locally (e.g., intratumorally) or systemically (e.g., intravenously). Administration can be used to treat subjects who have been confirmed to have a recognized condition, disease, or morbidity, or to treat subjects who are susceptible to or at risk of such a condition, disease, or morbidity. Co-administration with adjuvant therapy can include simultaneous and / or sequential delivery of multiple agents (e.g., modified immune cells with one or more cytokines; immunosuppressive therapies, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low doses of mycophenolic acid prodrugs, or any combination thereof) in any order and on any dosing schedule.

[0340] In certain embodiments, multiple doses of a composition described herein are administered to a subject, which compositions may be administered at an administration interval of about two weeks to about four weeks.

[0341] Treatment or prevention methods disclosed herein can be applied to a subject as a part of a course of treatment or scheme, and it can be included in the other treatment before or after the unit dose, cell or composition disclosed herein are applied.For example, in certain embodiments, the subject receiving the modified immune cell of a unit dose is receiving or has previously received hematopoietic cell transplantation (HCT; Including myeloablative and non-myeloablative HCT).Techniques and schemes for carrying out HCT are known in the art, and the transplantation of any suitable donor cell can be included, such as cells derived from cord blood, bone marrow or peripheral blood, hematopoietic stem cells, mobilized stem cells or cells from amniotic fluid.Therefore, in certain embodiments, the modified immune cell of the present disclosure can be applied soon after modified HCT therapy together with hematopoietic stem cells or after hematopoietic stem cells.In certain embodiments, HCT includes donor hematopoietic cells, and the donor hematopoietic cells include the chromosome knockout of the gene encoding HLA components, the gene chromosome knockout of encoding TCR components, or both.

[0342] In further embodiments, the subject has 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.

[0343] The method according to the present disclosure may further include administering one or more additional agents in combination therapy to treat a disease or condition. For example, in certain embodiments, combination therapy includes administering a composition of the present disclosure together with an immune checkpoint inhibitor (simultaneously, concurrently, or sequentially). In certain embodiments, combination therapy includes administering a composition of the present disclosure together with an agonist of a stimulating immune checkpoint agent. In further embodiments, combination therapy includes administering a composition of the present disclosure together with a secondary therapy, such as a chemotherapeutic agent, radiotherapy, surgery, an antibody, or any combination thereof.

[0344] 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, immunosuppressive agents include molecules that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immunosuppression. Exemplary immunosuppressive agents for targeting (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.

[0345] Immunosuppressive agent inhibitors (also known as immune checkpoint inhibitors) can be compounds, antibodies, antibody fragments or fusion polypeptides (e.g., Fc fusions such as CTLA4-Fc or LAG3-Fc), antisense molecules, ribozymes or RNAi molecules, or low molecular weight organic molecules. In any of the embodiments disclosed herein, the method can comprise the compositions of the present disclosure, alone or in any combination, with one or more inhibitors of any of the following immunosuppressive components.

[0346] In certain embodiments, the compositions of the present disclosure are used in combination with a PD-1 inhibitor, such as a PD-1 specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly known as AMP-514), AMP-224, BMS-936558, or any combination thereof. In further embodiments, the compositions of the present 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 envisioned are cemiplimab; IBI-308; nivolumab plus relatlimab; BCD-100; camrelizumab; JS-001; spartalizumab; tislelizumab; AGEN-2034; BGBA-333 plus tislelizumab; CBT-501; and dotalimab. dostarlimab); durvalumab + MEDI-0680; JNJ-3283; pazopanib hydrochloride + pembrolizumab; pidilizumab; REGN-1979 + cemiplizumab; ABBV-181; ADUS-100 + spartalizumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; cemiplizumab + REGN-3767; CS-1003; G LS-010; LZM-009; MEDI-5752; MGD-013; PF-06801591; Sym-021; tislelizumab plus pamiparib; XmAb-20717; AK-112; ALPN-202; AM-0001; antibodies that antagonize PD-1 in Alzheimer's disease; BH-2922; BH-2941; BH-2950; BH-2954; biologics that antagonize CTLA-4 and PD-1 in solid tumors; bispecific monoclonal antibodies targeting PD-1 and LAG-3 for oncology; BLSM-101; CB-201; CB-213; CBT-103; CBT-107; cellular immunotherapy plus PD-1 inhibitor; CX-188; HAB-21; HEISCOIII-003; IKT-202; JTX-4014; MCLA-134; MD-402;mDX-400; MGD-019; monoclonal antibodies targeting PDCD1 in oncology; monoclonal antibodies targeting PD-1 in oncology; oncolytic viruses inhibiting PD-1 in oncology; OT-2; PD-1 antagonist plus ropeginterferon alfa-2b; PEGMP-7; PRS-332; RXI-762; STIA-1110; TSR-075; vaccines targeting HER2 and PD-1 in oncology; vaccines targeting PD-1 in oncology and autoimmune disorders; XmAb-23104; antisense oligonucleotides inhibiting PD-1 in oncology; AT-16201; bispecific monoclonal antibodies inhibiting PD-1 in oncology; IMM-1802; vaccines targeting PD-1 and PD-1 in solid and hematologic tumors. Monoclonal antibodies targeting CTLA-4; biosimilars to nivolumab; recombinant proteins that agonize CD278 and CD28 and antagonize PD-1 for oncology; recombinant proteins that agonize PD-1 for autoimmune and inflammatory disorders; SNA-01; SSI-361; YBL-006; AK-103; JY-034; AUR-012; BGB-108; drugs that inhibit PD-1, Gal-9, and TIM-3 for solid tumors; ENUM-244C8; ENUM-388D4; M EDI-0680; monoclonal antibodies that antagonize PD-1 in metastatic melanoma and metastatic lung cancer; monoclonal antibodies that inhibit PD-1 in oncology; monoclonal antibodies that target CTLA-4 and PD-1 in oncology; monoclonal antibodies that antagonize PD-1 in NSCLC; monoclonal antibodies that inhibit PD-1 and TIM-3 in oncology; monoclonal antibodies that inhibit PD-1 in oncology; recombinant proteins that inhibit PD-1 and VEGF-A in hematological malignancies and solid tumors; and monoclonal antibodies that antagonize PD-1 in oncology. Small molecules targeting PD-1; 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 antagonizing 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 for solid tumors.

[0347] 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.

[0348] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CTLA4. In specific embodiments, the compositions of the present disclosure are used in combination with CTLA4-specific antibodies or binding fragments thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.

[0349] 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 can be a scFv or a fusion protein thereof, such as described, for example, in Dangaj et al., Cancer Res. 73:4820, 2013, and in U.S. Pat. No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724A1 and WO 2013 / 025779A1.

[0350] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CD244.

[0351] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of BLTA, HVEM, CD160, or any combination thereof. Anti-CD-160 antibodies are described, for example, in PCT Publication No. WO 2010 / 084158.

[0352] In certain embodiments, the disclosed cell compositions are used in combination with an inhibitor of TIM3.

[0353] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of Gal9.

[0354] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of adenosine signaling, such as decoy adenosine receptors.

[0355] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of A2aR.

[0356] In certain embodiments, the compositions of the present disclosure are used in combination with a KIR inhibitor, such as lirilumab (BMS-986015).

[0357] In certain embodiments, the compositions of the present disclosure are used in combination with suppressive cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

[0358] In certain embodiments, the compositions of the present disclosure are used in combination with an IDO inhibitor, such as L-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochemistry 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; April 6-10, 2013), 1-methyl-tryptophan (1-MT)-tirapazamine, or any combination thereof.

[0359] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as N(ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronylhexanoic acid (ABH), S-(2-boronylethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0360] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0361] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of TIGIT (e.g., COM902 (Compugen, Toronto, Ontario Canada)), an inhibitor of CD155 (e.g., COM701 (Compugen)), or both.

[0362] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. For example, anti-PVRIG antibodies are described in PCT Publication No. WO 2016 / 134333. For example, anti-PVRL2 antibodies are described in PCT Publication No. WO 2017 / 021526.

[0363] In certain embodiments, the compositions of the present disclosure are used in combination with a LAIR1 inhibitor.

[0364] 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.

[0365] In certain embodiments, the compositions of the present disclosure are used in combination with agents that increase the activity of stimulatory immune checkpoint molecules (ie, agonists). For example, the compositions of the present disclosure can be combined with 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 (e.g., CP-870,893, rhuCD40L, or SGN-40), CD122 agonists (e.g., IL-2), GITR agonists (e.g., humanized monoclonal antibodies described in PCT Patent Publication No. WO 2016 / 054638), ICOS (CD278) agonists (e.g., GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos314-8, or any combination thereof). In any of the embodiments disclosed herein, the method may comprise administering the compositions of the present disclosure alone or in any combination with one or more agonists of stimulatory immune checkpoint molecules, including any of the foregoing.

[0366] In certain embodiments, the combination therapy comprises a composition of the present disclosure and a secondary 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.

[0367] In certain embodiments, the combination therapy method comprises administering a 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 of ordinary skill in the art.

[0368] In certain embodiments, the combination therapy method comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (such as folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (such as intercalators), and DNA repair inhibitors. Illustrative chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents, 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)); antiproliferative / antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epipodophyllotoxins, and cytotoxic agents. ophyllotoxin (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, ifosfamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP 16)); 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 incapacitates cells that are unable to synthesize their own asparagine); antiplatelet agents;Antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (dichloromethyldiethylamine, cyclophosphamide and its analogs, melphalan, chlorambucil), ethyleneimine and methylmelamines (altretinoin and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and its analogs, streptozotocin), triazene-dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methylamino pterin); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants antithrombin agents (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytics (such as tissue prothrombin activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anticoagulants; antisecretory agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus); 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 toxin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin perturbations.

[0369] Cytokines can be used to manipulate host immune responses for anticancer activity. See, for example, Floros and Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines that can be used to promote immune anticancer or antitumor 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 alone or in any combination with the compositions of the present disclosure.

[0370] Also provided herein are methods for regulating adoptive immunotherapy, wherein the method comprises administering a homologous compound of a safety switch protein to a subject that has previously received a modified host cell of the present disclosure comprising a heterologous polynucleotide encoding the safety switch protein in an amount effective to eliminate the previously administered modified host cell in the subject.

[0371] 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.

[0372] In still other aspects, methods for preparing compositions or unit doses of the present disclosure are provided. 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 adoptive transfer therapy (e.g., targeting cancer antigens).

[0373] In some embodiments, the method further comprises culturing the transduced host cells before aliquoting and selecting the transduced cells that have incorporated (i.e., expressed) the vector. In other embodiments, the method comprises amplifying the transduced host cells after culturing and selecting and before aliquoting. In any of the embodiments of the methods of the present invention, the prepared composition or unit dose can be frozen or cryopreserved for later use. According to the methods of the present invention, any suitable host cell can be used to prepare the composition or unit dose, 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 a host cell that is a CD8 + T cells, CD4 + T cells or both.

[0374] Also provided are any of the binding proteins, polynucleotides, expression vectors, host cells, host cell compositions, unit doses, and immunogenic polypeptides, which are administered alone or in any combination for treating a disease or condition associated with a KRAS G12D mutation, a KRAS G12V mutation, a NRAS G12D mutation, a NRAS G12V mutation, a HRAS G12V mutation, or a HRAS G12D mutation in a subject.

[0375] Also provided are any of the binding proteins, polynucleotides, expression vectors, host cells, host cell compositions, unit doses, and immunogenic polypeptides, which are taken alone or in any combination for the preparation of a medicament for treating a disease or condition associated with a KRASG12D mutation, or a KRAS G12V, or a NRAS G12D mutation, or a NRAS G12V mutation, or a HRAS G12V mutation, or a HRAS G12D mutation in a subject.

[0376] In certain embodiments, the disease or condition comprises cancer. In some embodiments, the cancer is a solid cancer or a hematological malignancy. In certain embodiments, the disease or condition is selected from pancreatic cancer (pancreas cancer or pancreas carcinoma), optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer (colorectal cancer or colorectal carcinoma); lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer (endometrial cancer or endometrial 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 lymphoblastic leukemia (ALL); urinary tract cancer; small intestinal cancer; breast cancer (breast cancer or breast carcinoma); melanoma (optionally cutaneous melanoma, anal melanoma, or mucosal melanoma); glioma; poorly differentiated thyroid carcinoma; neuroblastoma; histiocytic and dendritic cell neoplasms; neurofibromatosis type 1; rhabdomyosarcoma; soft tissue sarcoma; bladder cancer carcinoma); 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 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; thyroid malignant neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma or papillary thyroid carcinoma. In some embodiments, the method comprises administering a subject composition parenterally or intravenously. In some embodiments, the method comprises administering multiple doses of a binding protein, polynucleotide, expression vector, host cell, host cell composition, unit dose, and / or immunogenic polypeptide to a subject.

[0377] In certain embodiments, the multiple doses are administered at an administration interval of about two weeks to about four weeks.

[0378] In certain embodiments, the composition comprises a modified host cell. In some embodiments, the method comprises administering to about 10 4 cells / kg to about 10 11 The modified host cells are administered to a subject at a dose of 10 cells / kg.

[0379] In certain embodiments, wherein the method further comprises administering a cytokine to the subject. In some embodiments, the cytokine comprises IL-2, IL-15, or IL-21.

[0380] In certain embodiments, the subject has received or is currently receiving an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

[0381] Also provided are methods comprising introducing a polynucleotide encoding a binding protein of the disclosure into a host (eg, T) cell.

[0382] The present disclosure also provides the following non-limiting examples.

[0383] Example 1. A binding protein comprising:

[0384] (a) a T cell receptor (TCR) alpha 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

[0385] (b) a TCR β chain variable (Vβ) domain comprising the CDR3β amino acid sequence shown 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,

[0386] 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 wherein the HLA comprises HLA-A * 11.

[0387] Example 2. The binding protein according to Example 1, wherein the HLA comprises HLA-A * 11:01.

[0388] Embodiment 3. The binding protein according to embodiment 1 or 2, wherein the Vα domain and / or the Vβ domain is human, humanized or chimeric, and preferably human.

[0389] Embodiment 4. The binding protein according to any one of embodiments 1 to 3, comprising the CDR3α and CDR3β amino acid sequences shown below: (i) SEQ ID NOs.: 17 and 27, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; (ii) SEQ ID NOs.: 16 and 26, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; (iii) SEQ ID NOs.: 53 and 43, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; or (iv) SEQ ID NOs.: 52 and 42, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions.

[0390] Embodiment 5. The binding protein according to any one of embodiments 1 to 4, comprising: (i) in the Vα domain, the CDR1α amino acid sequence shown in SEQ ID NO.: 14 or 40, or a variant thereof having one or two optionally conservative amino acid substitutions; (ii) in the Vα domain, the CDR2α amino acid sequence shown in SEQ ID NO.: 15 or 41, or a variant thereof having one or two optionally conservative amino acid substitutions; (iii) in the Vβ domain, the CDR1β amino acid sequence shown in SEQ ID NO.: 24 or 50, or a variant thereof having one or two optionally conservative amino acid substitutions; (iv) in the Vβ domain, the CDR2β amino acid sequence shown in SEQ ID NO.: 25 or 51, or a variant thereof having one or two optionally conservative amino acid substitutions; or any combination of (v) (i)-(iv).

[0391] Embodiment 6. The binding protein according to any one of embodiments 1 to 5, comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences shown in SEQ ID NO.: 14, 15, 16 or 17, 24, 25 and 26 or 27, respectively.

[0392] Embodiment 7. The binding protein according to any one of embodiments 1 to 5, comprising the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences shown in SEQ ID NO.: 40, 41, 42 or 43, 50, 51 and 52 or 53, respectively.

[0393] Embodiment 8. The binding protein according to any one of embodiments 1 to 7, wherein:

[0394] (i) the Va domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 13 or 39; and / or

[0395] (ii) the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 23 or 49.

[0396] Embodiment 9. The binding protein of any one of embodiments 1 to 8, wherein the Vα domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 13, and wherein the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 23, wherein, optionally, the binding protein comprises SEQ ID NO. The amino acid sequence shown in NO.:154.

[0397] Embodiment 10. The binding protein of any one of embodiments 1 to 8, wherein the Vα domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 39, and wherein the Vβ domain comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 49.

[0398] Embodiment 11. The binding protein of any one of embodiments 1 to 10, wherein the Vα domain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO.: 13, and the Vβ domain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO.: 23, wherein, optionally, the binding protein comprises the amino acid sequence shown in SEQ ID NO.: 154.

[0399] Embodiment 12. The binding protein of any one of embodiments 1 to 10, wherein the Vα domain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO.: 39, and the Vβ domain comprises, consists essentially of, or consists of the amino acid sequence shown in SEQ ID NO.: 49.

[0400] Embodiment 13. The binding protein according to any one of embodiments 1 to 12, further comprising a TCR alpha chain constant domain (Ca) and / or a TCR beta chain constant domain (Cβ).

[0401] Embodiment 14. The binding protein of embodiment 13, wherein the Cα comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in any one of SEQ ID NO.: 18, 19, 44, 45 and 69.

[0402] Embodiment 15. The binding protein of embodiment 13 or 14, wherein the Cβ comprises, consists essentially of, or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs.: 28, 29, 54, 55, and 70-73.

[0403] Embodiment 16. The binding protein of any one of embodiments 13 to 15, wherein said Cα and said Cβ comprise or consist of an amino acid sequence that is 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% identical to the amino acid sequence shown in:

[0404] (i) SEQ ID NO.: 18 and 28, respectively;

[0405] (ii) SEQ ID NO.: 19 and 29, respectively;

[0406] (iii) SEQ ID NO.: 44 and 54, respectively; or

[0407] (iv) SEQ ID NO.: 45 and 55, respectively.

[0408] Embodiment 17. The binding protein of any one of embodiments 1 to 16, comprising a TCR alpha chain and a TCR beta chain, wherein said TCR alpha chain and said TCR beta chain comprise or consist of an amino acid sequence that is 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% identical to the amino acid sequence shown in:

[0409] (i) SEQ ID NO.: 12 and 22, respectively;

[0410] (ii) SEQ ID NO.: 20 and 30 or 155, respectively;

[0411] (iii) SEQ ID NO.: 12 and 30 or 155, respectively;

[0412] (iv) SEQ ID NOs.: 20 and 22, respectively;

[0413] (v) SEQ ID NOs.: 38 and 48, respectively;

[0414] (vi) SEQ ID NOs.: 46 and 56, respectively;

[0415] (vii) SEQ ID NO.: 38 and 56, respectively; or

[0416] (viii) SEQ ID NO.: 46 and 48, respectively.

[0417] Embodiment 18. The binding protein of any one of embodiments 1 to 17, 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).

[0418] Example 19. The binding protein of Example 18, wherein the binding protein comprises a TCR.

[0419] Embodiment 20. An isolated polynucleotide encoding the binding protein of any one of embodiments 1 to 19.

[0420] Embodiment 21. A polynucleotide according to embodiment 20, comprising or consisting of a polynucleotide 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 sequence shown in any one of SEQ ID NOs.: 5-10 and 33-36, or any combination thereof.

[0421] Embodiment 22. The polynucleotide of embodiment 20 or 21, further comprising:

[0422] (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor alpha chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor alpha chain;

[0423] (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or

[0424] (iii) the polynucleotide of (i) and the polynucleotide of (ii).

[0425] Embodiment 23. The polynucleotide of embodiment 22, comprising:

[0426] (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor α chain;

[0427] (b) the polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain; and

[0428] (c) A polynucleotide encoding a self-cleaving peptide, the polynucleotide being positioned between the polynucleotide of (a) and the polynucleotide of (b).

[0429] Embodiment 24. The polynucleotide of embodiment 22 or 23, further comprising a polynucleotide encoding a self-cleaving peptide positioned between:

[0430] (1) the polynucleotide encoding the binding protein and the polynucleotide encoding the polypeptide, wherein the polypeptide comprises the extracellular portion of the CD8 co-receptor α chain; and / or

[0431] (2) The polynucleotide encoding the binding protein and the polynucleotide encoding the polypeptide, wherein the polypeptide comprises the extracellular portion of the CD8 co-receptor β chain.

[0432] Embodiment 25. The polynucleotide of any one of embodiments 22 to 24, comprising operably linked in frame:

[0433] (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnBP);

[0434] (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnBP);

[0435] (iii) (pnBP)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β);

[0436] (iv) (pnBP)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α);

[0437] (v) (pnCD8α)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8β); or

[0438] (vi)(pnCD8β)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8α),

[0439] wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain,

[0440] wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain,

[0441] wherein pnBP is a polynucleotide encoding a binding protein,

[0442] And wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0443] Embodiment 26. The polynucleotide of any one of embodiments 22 to 25, wherein the encoded binding protein comprises a TCR alpha chain and a TCR beta chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide positioned between a polynucleotide encoding the TCR alpha chain and a polynucleotide encoding the TCR beta chain.

[0444] Embodiment 27. The polynucleotide of embodiment 26, comprising operably linked in frame:

[0445] (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα);

[0446] (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα);

[0447] (iii) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ);

[0448] (iv) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ);

[0449] (v) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β);

[0450] (vi) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α);

[0451] (vii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β);

[0452] (viii)(pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α),

[0453] wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain,

[0454] wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain,

[0455] wherein pnTCRα is the polynucleotide encoding the TCRα chain,

[0456] wherein pnTCRβ is the polynucleotide encoding the TCRβ chain,

[0457] And wherein pnSCP1, pnSCP2 and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

[0458] Embodiment 28. The polynucleotide of any one of embodiments 20 to 27, which encodes an amino acid sequence that is 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% identical to the amino acid sequence shown in any one of SEQ ID NO.: 11, 21, 37, 47, 31, 32, 57 and 58, or comprises or consists of the amino acid sequence shown in any one of them.

[0459] Embodiment 29. The polynucleotide of embodiment 28, encoding (i) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 11, or comprises or consists of the amino acid sequence shown therein, and (ii) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 21, or comprises or consists of the amino acid sequence shown therein.

[0460] Embodiment 30. The polynucleotide of embodiment 29, which encodes (i) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: ...

Claims

1. A binding protein comprising: (a) a T cell receptor (TCR) alpha chain variable (Vα) domain comprising the complementarity determining region 3 (CDR3α) amino acid sequence shown 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 the CDR3β amino acid sequence shown 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, wherein the binding protein is capable of binding to a peptide:HLA complex, wherein the peptide comprises or consists of the amino acid sequence VVVGAVGVGK (SEQ ID NO.: 2) or VVGAVGVGK (SEQ ID NO.: 3), and wherein the HLA comprises HLA-A * 11.

2. The binding protein according to claim 1, wherein the HLA comprises HLA-A * 11:

01. 3 . The binding protein according to claim 1 , wherein the Vα domain and / or the Vβ domain is human, humanized or chimeric, and preferably human.

4. The binding protein according to any one of claims 1 to 3, comprising the CDR3α and CDR3β amino acid sequences shown below: (i) SEQ ID NOs.: 17 and 27, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; (ii) SEQ ID NOs.: 16 and 26, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; (iii) SEQ ID NOs.: 53 and 43, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions; or (iv) SEQ ID NOs.: 52 and 42, respectively, or variants thereof having one, two or three optionally conservative amino acid substitutions.

5. The binding protein according to any one of claims 1 to 4, comprising: (i) in the Vα domain, the CDR1α amino acid sequence shown in SEQ ID NO.: 14 or 40, or a variant thereof having one or two optionally conservative amino acid substitutions; (ii) in the Vα domain, the CDR2α amino acid sequence shown in SEQ ID NO.: 15 or 41, or a variant thereof having one or two optionally conservative amino acid substitutions; (iii) in the Vβ domain, the CDR1β acid sequence shown in SEQ ID NO.: 24 or 50, or a variant thereof having one or two optionally conservative amino acid substitutions; (iv) in the Vβ domain, the CDR2β acid sequence shown in SEQ ID NO.: 25 or 51, or a variant thereof having one or two optionally conservative amino acid substitutions; or any combination of (v) (i)-(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 shown 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 shown in SEQ ID NO.: 40, 41, 42 or 43, 50, 51 and 52 or 53, respectively.

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

9. The binding protein of any one of claims 1 to 8, wherein the Vα domain comprises or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 13, and wherein the Vβ domain comprises or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.:

23.

10. The binding protein of any one of claims 1 to 8, wherein the Vα domain comprises or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 39, and wherein the Vβ domain comprises or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.:

49.

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

23.

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

49. 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 binding protein of claim 13, wherein the Cα comprises or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in any one of SEQ ID NO.: 18, 19, 44, 45 and 69, or comprises or consists of the amino acid sequence shown in any one of them.

15. The binding protein according to claim 13 or 14, wherein the Cβ comprises or consists of an amino acid sequence that is 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% identical to the amino acid sequence shown in any one of SEQ ID NOs.: 28, 29, 54, 55 and 70-73, or comprises or consists of the amino acid sequence shown in any one of them.

16. The binding protein according to any one of claims 13 to 15, wherein the Ca and the Cβ comprise or consist of an amino acid sequence that is 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% identical to the amino acid sequence shown in: (i) SEQ ID NO.: 18 and 28, respectively; (ii) SEQ ID NO.: 19 and 29, respectively; (iii) SEQ ID NO.: 44 and 54, respectively; or (iv) SEQ ID NO.: 45 and 55, respectively. 17 . The binding protein of any one of claims 13 to 16 , wherein the Ca, the Cβ, or both comprise modifications that promote preferential pairing of the Ca with the Cβ.

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

19. The binding protein of any one of claims 13 to 16, wherein the Ca comprises a T48C substitution and the Cβ comprises a S57C substitution to promote preferential pairing of the Ca with the Cβ.

20. The binding protein according to any one of claims 1 to 19, comprising a TCR alpha chain and a TCR beta chain, wherein the TCR alpha chain and the TCR beta chain comprise or consist of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the amino acid sequence shown in: (i) SEQ ID NO.: 12 and 22, respectively; (ii) SEQ ID NO.: 20 and 30, respectively; (iii) SEQ ID NO.: 12 and 30, respectively; (iv) SEQ ID NO.: 20 and 22, respectively; (v) SEQ ID NOs.: 38 and 48, respectively; (vi) SEQ ID NOs.: 46 and 56, respectively; (vii) SEQ ID NO.: 38 and 56, respectively; or (viii) are SEQ ID NOs.: 46 and 48, respectively.

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 of claim 21, wherein the binding protein comprises a TCR.

23. The binding protein of any one of claims 1 to 22, wherein the functional affinity of the binding protein for the peptide in a CD137 surface expression assay comprises an EC50 of at most 100 nM, at most 50 nM, at most 25 nM, at most 10 nM, at most 1 nM, at most 750 pM, at most 500 pM, at most 250 pM, at most 100 pM, at most 75 pM or at most 60 pM.

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

25. A polynucleotide according to claim 24, comprising a polynucleotide 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 a polynucleotide sequence shown in any one of SEQ ID NOs.: 5-10 and 33-36, or any combination thereof, or comprising or consisting of a polynucleotide sequence shown in any one of them, or any combination thereof.

26. The polynucleotide according to claim 24 or 25, further comprising: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the α chain of the CD8 co-receptor, wherein Optionally, the encoded polypeptide is or comprises a CD8 co-receptor alpha chain; (ii) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii).

27. The polynucleotide according to claim 26, comprising: (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of the α chain of the CD8 co-receptor; (b) the polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain; and (c) a polynucleotide encoding a self-cleaving peptide, the polynucleotide being disposed between the polynucleotide of (a) and the polynucleotide of (b).

28. The polynucleotide of claim 26 or 27, further comprising a polynucleotide encoding a self-cleaving peptide disposed between: (1) the polynucleotide encoding the binding protein and the polynucleotide encoding the polypeptide, wherein the polypeptide comprises the extracellular portion of the CD8 co-receptor α chain; and / or (2) The polynucleotide encoding the binding protein and the polynucleotide encoding the polypeptide, wherein the polypeptide comprises the extracellular portion of the CD8 co-receptor β chain.

29. A polynucleotide according to any one of claims 26 to 28, comprising 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)-(pnSCP1)-(pnBP)-(pnSCP2)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnBP is a polynucleotide encoding a binding protein, And wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

30. The polynucleotide of any one of claims 26 to 29, wherein the encoded binding protein comprises a TCR alpha chain and a TCR beta chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between a polynucleotide encoding the TCR alpha chain and a polynucleotide encoding the TCR beta chain.

31. The polynucleotide of claim 30, comprising operably linked in frame: (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)-(pnSCP3)-(pnCD8α); (vii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β); (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, wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnTCRα is the polynucleotide encoding the TCRα chain, wherein pnTCRβ is the polynucleotide encoding the TCRβ chain, And wherein pnSCP1, pnSCP2 and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different.

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

33. A polynucleotide according to any one of claims 24 to 32, encoding an amino acid sequence that is 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% identical to the amino acid sequence shown in any one of SEQ ID NO.: 11, 21, 37, 47, 31, 32, 57 and 58, or comprises or consists of the amino acid sequence shown in any one of them.

34. The polynucleotide of claim 33, encoding (i) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 11, or comprises or consists of the amino acid sequence shown therein, and (ii) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 21, or comprises or consists of the amino acid sequence shown therein.

35. The polynucleotide of claim 33, encoding (i) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 37, or comprises or consists of the amino acid sequence shown therein, and (ii) an amino acid sequence that is 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% identical to the amino acid sequence shown in SEQ ID NO.: 47, or comprises or consists of the amino acid sequence shown therein.

36. A polynucleotide according to any one of claims 24 to 35, which is or comprises a polynucleotide sequence which is codon-optimized for expression in a host cell, wherein Optionally, the host cell is a human immune system cell, and further optionally 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 of claim 37, wherein the expression control sequence comprises an MSCV promoter.

39. An expression vector according to claim 37 or claim 38, wherein the expression control sequence drives 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 TCR α chain and the TCR β chain.

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

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

42. The expression vector of claim 41, wherein the human immune system cell is a CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, gamma delta T cells, natural killer cells, natural killer T cells, macrophages, monocytes, dendritic cells, or any combination thereof.

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

45. The expression vector of claim 44, wherein the viral vector is a lentiviral vector or a gammaretroviral vector.

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

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

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

49. The host cell of claim 48, wherein the modified cells comprise hematopoietic progenitor cells and / or human immune cells.

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

51. The host cell of claim 50, wherein the immune cell comprises a 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, in, Optionally, the immune cells comprise CD4 + T cells and CD8 + T cells, wherein, further optionally, the CD4 + T cells, CD8 + T cells, or both, comprise (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (ii) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

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

53. The host cell of claim 52, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from the group consisting of an α1 macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a β1 microglobulin gene, or a β2 microglobulin gene.

54. The host cell of claim 52 or 53, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from the group consisting of a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or a combination thereof.

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

56. The composition of claim 55, comprising at least about 30% modified CD4 + T cells, the composition and (ii) comprising at least about 30% of modified CD8 + The composition of T cells is combined at a ratio of about 1:

1.

57. The composition of claim 55 or 56, wherein the composition is substantially free of 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) the expression vector according to any one of claims 37 to 47; and / or (xii) the host cell according to any one of claims 48 to 54, and a pharmaceutically acceptable carrier, excipient or diluent.

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

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

61. The method of claim 59 or 60, wherein the disease or disorder is selected from pancreatic cancer (pancreascancer or pancreas carcinoma), optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer (colorectal cancer or colorectal carcinoma); lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer (endometrial cancer or endometrial 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 lymphocytic leukemia (ALL); urethral cancer; small intestine cancer; breast cancer (breast 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 carcinoma; 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 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; neurothecoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; thyroid malignant neoplasm; thyroid cancer; thyroid adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; papillary thyroid carcinoma; or their advanced or metastatic versions.

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

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

64. The method of claim 63, wherein the multiple doses are administered at an administration interval of about two weeks to about four weeks.

65. according to the method described in any one of claims 59 to 64, wherein said composition comprises said host cell or said composition comprising said host cell, and wherein said method comprises about 10 4 cells / kg to about 10 11 The host cell or the composition is administered to the subject at a dose of 1 cell / kg.

66. according to the method described in any one of claims 59 to 65, wherein said method comprises administering at least 5x 10^8, at least 1x 10^9, at least 5x10^9, at least 1x10^10, at least 1.5x 10^10, at least 2x 10^10 or at least 5x 10^10 live host cells comprising said binding protein to said subject, optionally in a single dose.

67. The method of any one of claims 59 to 65, wherein the method comprises administering to the subject, optionally in a single dose, at most 5 x 10^9, at most 1 x 10^10, at most 1.5 x 10^10, at most 2 x 10^10, at most 5 x 10^10, at most 1 x 10^11, or at most 5 x 10^11 live host cells comprising the binding protein.

68. according to the method described in any one of claims 59 to 65, wherein said method comprises administering about 5x 10^9, about 6x 10^9, about 7x 10^9, about 8x 10^9, about 9x 10^9, about 1x 10^10, about 1.1x 10^10, about 1.2x 10^10, about 1.3x 10^10, about 1.4x 10^10, about 1.5x10^10, about 1.6x 10^10, about 1.7x 10^10, about 1.8x 10^10, about 1.9x 10^10 or about 2x 10^10 live host cells comprising said binding protein to said subject, optionally in a single dose.

69. according to the method described in any one of claims 59 to 65, wherein the method comprises administering to the subject about 5x 10^9 to about 1x 10^11, about 5x 10^9 to about 5x 10^11, about 5x 10^9 to about 5x 10^11, about 5x 10^9 to about 2x 10^10, about 5x 10^9 to about 1.5x 10^10, about 5x 10^9 to about 1x 10^10, about 1x 10^10 to about 1x 10^11, about 1x 10^10 to about 5x 10^10, about 1x 10^10 to about 2x 10^10, or about 1x 10^10 to about 1.5x 10^10 live host cells comprising the binding protein, optionally in a single dose.

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

01.

71. The method of any one of claims 59 to 70, wherein the method further comprises administering a cytokine to the subject.

72. The method of claim 71, wherein the cytokine comprises IL-2, IL-15, or IL-21.

73. The method of 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.

74. 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, wherein, Optionally, the host cell comprises CD8+ T cells, CD4+ T cells or both, and / or the composition according to any one of claims 55 to 58, which is used in a method for treating a disease or condition associated with a KRAS G12V or NRASG12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or condition comprises cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or condition is selected from pancreatic cancer (pancreas cancer or pancreas carcinoma), optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer (colorectal cancer or colorectal carcinoma); lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer (endometrial carcinoma); cervical cancer; ovarian cancer; bladder 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 lymphoblastic leukemia (ALL); urethral cancer; small intestine cancer; breast cancer (breast cancer or breast 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 (bladder carcinoma); 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 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; neurothecoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; thyroid malignant neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; papillary thyroid carcinoma; or their advanced or metastatic versions.

75. 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, wherein, Optionally, the host cell comprises CD8+T cells, CD4+T cells or both, and / or the composition according to any one of claims 55 to 58, which is used in the preparation of a medicament for treating a disease or condition associated with a KRAS G12V or NRAS G12V mutation or a HRAS G12V mutation in a subject, wherein, optionally, the disease or condition comprises cancer, wherein, further optionally, the cancer is a solid cancer or a hematological malignancy, and wherein, optionally, the disease or condition is selected from pancreatic cancer (pancreas cancer or pancreas carcinoma), optionally pancreatic ductal adenocarcinoma (PDAC); colorectal cancer (colorectal cancer or colorectal carcinoma); lung cancer, optionally non-small cell lung cancer; biliary tract cancer; endometrial cancer (endometrial carcinoma); cervical cancer; ovarian cancer; bladder 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 lymphoblastic leukemia (ALL); urethral cancer; small intestine cancer; breast cancer; 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 (bladder cancer); carcinoma); 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 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; neurothecoma; secondary AML; small cell lung cancer; therapy-related AML; thymic carcinoma; follicular thyroid carcinoma; thyroid malignant neoplasm; thyroid carcinoma; thyroid adenocarcinoma; urothelial carcinoma; colon cancer; colorectal adenocarcinoma; papillary thyroid carcinoma; or their advanced or metastatic versions.

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