HLA class II-restricted T cell receptor for RAS with the G12V mutation

Abstract

Disclosed are isolated or purified T cell receptors (TCRs) having antigen specificity for mutant RAS amino acid sequences presented by human leukocyte antigen (HLA) class II molecules. Related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions, are also provided. Also disclosed are methods for detecting the presence of cancer in a mammal and for treating or preventing cancer in a mammal.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 560,930, filed February 26, 2020, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Project No. ZIABC010984 by the National Cancer Institute, National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Submitted Documents The computer-readable nucleotide / amino acid sequence listing, submitted concurrently herewith and identified as follows, is incorporated herein by reference in its entirety: One ASCII (text) file of 266,276 bytes entitled "751507_ST25.txt", dated February 18, 2021. [Background technology]

[0004] Some cancers may have very limited treatment options, especially when the cancer becomes metastatic and unresectable. For example, despite advances in treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may be poor. Thus, there is an unmet need for additional cancer treatments. Summary of the Invention

[0005] An embodiment of the present invention provides an isolated or purified T cell receptor (TCR) comprising the amino acid sequence of (a) SEQ ID NOs: 1 to 3, (b) SEQ ID NOs: 4 to 6, (c) SEQ ID NOs: 31 to 33, (d) SEQ ID NOs: 34 to 36, (e) SEQ ID NOs: 1 to 6, or (f) SEQ ID NOs: 31 to 36, wherein the TCR has antigen specificity for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule, in which glycine at position 12 is substituted with valine, and the mutant human RAS amino acid sequence is the amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS), and wherein position 12 is defined by reference to the wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively.

[0006] Another embodiment of the present invention provides an isolated or purified polypeptide comprising a functional portion of a TCR of the present invention, wherein the functional portion comprises the amino acid sequence of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 31 to 33, (d) all of SEQ ID NOs: 34 to 36, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 31 to 36.

[0007] Yet another embodiment of the present invention provides an isolated or purified protein comprising at least one of the polypeptides of the present invention.

[0008] Further embodiments of the present invention provide nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions related to the TCRs, polypeptides, and proteins of the present invention.

[0009] An embodiment of the present invention is an isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences are set forth, respectively, as SEQ ID NOs: 7 and 8; 7 and 64; 63 and 8; 63 and 64; 7 and 65; 63 and 65; 7 and 66; 63 and 66; 8 and 7; 64 and 7; 8 and 63; 64 and 63; 65 and 7; 65 and 63; 66 and 7; 66 and 63; 129 and 8; 129 and 64; 129 and 65; 129 and 66; 8 and 129; 64 and 129; 65 and 129; 66 and 1 29;130 and 8;130 and 64;130 and 65;130 and 66;8 and 130;64 and 130;65 and 130;66 and 130;37 and 38;37 and 69;37 and 70;37 and 71;38 and 37;69 and 37;70 and 37;71 and 37;23 and 24;23 and 84;83 and 24;83 and 84;23 and 87;83 and 87;23 and 90;83 and 90;24 and 23;84 and 23;24 and 83;84 and 83;87 and 23;87 and 83;90 and 23;90 and 83;133 and 24;133 and 84; 133 and 87;133 and 90;24 and 133;84 and 133;87 and 133;90 and 133;39 and 40;39 and 107;39 and 112;39 and 115;40 and 39;107 and 39;112 and 39;115 and 39;136 and 24;136 and 84;136 and 87;136 and 90;24 and 136;84 and 136;87 and 136;90 and 136;21 and 22;21 and 80;79 and 22;79 and 80;21 and 85;21 and 88;79 and 85;79 and 88;22 and 21;80 and 21;22 and 7 9;80 and 79;85 and 21;88 and 21;85 and 79;88 and 79;131 and 22;131 and 80;131 and 85;131 and 88;22 and 131;80 and 131;85 and 131;88 and 131;134 and 22;134 and 80;134 and 85;134 and 88;22 and 134;80 and 134;85 and 134;88 and 134;77 and 78;77 and 82;81 and 78;81 and 82;77 and 86;81 and 86;78 and 77;82 and 77;78 and 81;82 and 81;86 and 77;86 and 81;132 and 78;132 and 82;132 and 86;78 and 132;82 and 132;86 and 132;135 and 78;135 and 82;135 and 86;78 and 135;82 and 135;86 and 135;77 and 89;81 and 89;89 and 77;89 and 81;132 and 89;89 and 132;135 and 89;89 and 135;41 and 42;41 and 105; 41 and 110;41 and 113;42 and 41;105 and 41;110 and 41;113 and 41;103 and 104;103 and 111;103 and 114;104 and 103;111 and 103;114 and 103;103 and 106;106 and 103;47 and 48;48 and 47;67 and 68;67 and 76;68 and 67;76 and 67;49 and 50;50 and and 49;72 and 73;72 and 102;73 and 72;102 and 72;51 and 52;52 and 51;53 and 54;54 and 53;55 and 56;56 and 55;57 and 58;58 and 57;91 and 92;92 and 91;108 and 109;109 and 108;93 and 94;93 and 99;94 and 93;99 and 93;97 and 98;97 and 101;98 and 97 Nucleic acids encoding the amino acid sequences of: 101 and 97; 95 and 96; 95 and 100; 96 and 95; 100 and 95; 116 and 117; 116 and 122; 117 and 116; 122 and 116; 120 and 121; 120 and 124; 121 and 120; 124 and 120; 118 and 119; 118 and 123; 119 and 118, or 123 and 118 are provided.

[0010] Further provided by embodiments of the invention are methods of detecting the presence of cancer in a mammal, methods of treating or preventing cancer in a mammal, methods of inducing an immune response against cancer in a mammal, methods of producing host cells expressing a TCR with antigen specificity for the peptide of SEQ ID NO: 30, and methods of producing the TCRs, polypeptides, and proteins of the invention.

[0011] Additional embodiments are as described herein. [Brief explanation of the drawings]

[0012] [Figure 1A] Flow cytometry dot plots are presented showing cell sorting during an in vitro stimulation (IVS) protocol to select T cells for expansion. REP is a rapid expansion protocol. [Figure 1B] Flow cytometry dot plots are presented showing cell sorting during the in vitro stimulation (IVS) protocol after selection and expansion of T cells shown in Figure 1A. [Figure 1C] FIG. 1C is a graph showing the results of IFN-γ ELISpot analysis on cells sorted as shown in FIG. 1B. [Figure 1D] FIG. 1C is a graph showing the results of an analysis of the upregulation of 41BB / OX40 surface markers in cells sorted as shown in FIG. 1B. [Figure 2A] 10 is a graph showing the results of IFN-γ ELISpot analysis of cells co-cultured with DCs loaded with RASG12VLP or RASWTLP. [Figure 2B] Figure 10 is a graph showing the results of an analysis of the upregulation of 41BB / OX40 surface markers in cells co-cultured with DCs loaded with RASG12VLP or RASWTLP. [Figure 3] Graphs showing the results of IFN-γ ELISpot and 41BB / OX40 flow cytometry assays used to identify MHC-II restriction elements recognized by TCR. [Figure 4A] 1 is a bar graph showing luciferase activity measured on Jurkat-CD4-NFAT-luciferase cell lines and then co-cultured with DCs loaded with RASG12V, RASWTLP, or an equivalent amount of DMSO. [Figure 4B-4C] Graphs showing TCR reactivity assessed by flow cytometry assay for 4-1BB and OX40 (%4-1BB / OX40) expression in CD3 / CD8 gated cells (FIG. 4B) or CD3 / CD4 gated cells (FIG. 4C). (-) indicates non-transduced cells. [Figure 5A]Graphs showing the measurement of 41BB and OX40 expression by flow cytometry of TILs of the indicated fractions stimulated by IVS and cocultured with autologous DCs pulsed with RASG12VLP peptide or transfected with RASG12VFL RNA. Negative controls: T cells cocultured alone, PBLs cultured with DMSO-loaded DCs. Positive controls: PBLs cultured with anti-CD3 / anti-CD28 antibody-conjugated Dynabeads. * indicates cell pooling. [Figure 5B] The results of IFN-γ ELISpot used to identify MHC-II restriction elements recognized by TILs are presented. [Figure 6] IFN-γ ELISpot results of TCR65-10 compared to TCR1 are presented. [Figures 7A-7D] Graphs are presented showing the results of flow cytometry assays of 4-1BB and OX40 (%4-1BB+ / OX40+) expression in TCR1-transduced PBLs gated on CD4 (Figure 7A) or CD8 (Figure 7B), and TCR5-transduced PBLs gated on CD4 (Figure 7C) or CD8 (Figure 7D). [Figures 7E-7G] Graphs are presented showing the results of ELISPOT assays of IFN-γ secretion levels in TCR1-transduced PBLs enriched for CD4 cells (Figure 7E) or CD8 cells (Figure 7F) and TCR5-transduced PBLs separated into CD4 cells or CD8 cells (Figure 7G). DETAILED DESCRIPTION OF THE INVENTION

[0013] RAS family proteins belong to a large family of small GTPases. Without being bound by any particular theory or mechanism, it is believed that when mutated, RAS proteins may be involved in signal transduction during the early stages of carcinogenesis in many human cancers. A single amino acid substitution can activate the protein. Mutant RAS protein products can be constitutively activated. Mutant RAS proteins can be expressed in any of a variety of human cancers, such as pancreatic cancer (e.g., pancreatic carcinoma), colorectal cancer, lung cancer (e.g., lung adenocarcinoma), endometrial cancer, ovarian cancer (e.g., epithelial ovarian cancer), and prostate cancer. Human RAS family proteins include Kirsten rat sarcoma viral oncogene homolog (KRAS), Harvey rat sarcoma viral oncogene homolog (HRAS), and neuroblastoma rat sarcoma viral oncogene homolog (NRAS).

[0014] KRAS is also referred to as GTPase KRas, V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. Two transcript variants of KRAS exist: KRAS variant A and KRAS variant B. Wild-type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO: 9. Wild-type (WT) KRAS variant B has the amino acid sequence of SEQ ID NO: 10. Hereinafter, reference to "KRAS" (mutated or non-mutated (WT)) refers to both variant A and variant B unless otherwise specified. When activated, mutant KRAS binds guanosine-5'-triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP).

[0015] HRAS is another member of the RAS protein family. HRAS is also known as Harvey rat sarcoma viral oncoprotein, V-Ha-Ras Harvey rat sarcoma viral oncogene homolog, or Ras family small GTP-binding protein H-Ras. WT HRAS has the amino acid sequence of SEQ ID NO: 11.

[0016] NRAS is another member of the RAS protein family. NRAS is also called GTPase NRas, V-Ras neuroblastoma RAS viral oncogene homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 12.

[0017] An embodiment of the present invention provides an isolated or purified TCR having antigen specificity for the amino acid sequence of mutant human RAS (hereinafter "mutant RAS") presented by human leukocyte antigen (HLA) class II molecules, in which glycine at position 12 is substituted with valine, wherein the amino acid sequence of the mutant human RAS is that of mutant human KRAS, mutant human HRAS, or mutant human NRAS, and position 12 is defined by reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. Hereinafter, reference to "TCR" also refers to functional portions and functional variants of the TCR, unless otherwise specified.

[0018] The TCRs of the present invention may have antigen specificity for any mutant human RAS protein, polypeptide, or peptide amino acid sequence. In an embodiment of the present invention, the mutant human RAS amino acid sequence is a mutant human KRAS amino acid sequence, a mutant human HRAS amino acid sequence, or a mutant human NRAS amino acid sequence. The amino acid sequences of wild-type human KRAS, NRAS, and HRAS proteins each have a length of 188 to 189 amino acid residues and are highly identical to each other. For example, the amino acid sequence of the wild-type human NRAS protein is 86.8% identical to the amino acid sequence of the wild-type human KRAS protein. Amino acid residues 1 to 86 of the wild-type human NRAS protein and the wild-type human KRAS protein are 100% identical. The amino acid sequence of the wild-type human HRAS protein is 86.3% identical to the amino acid sequence of the wild-type human KRAS protein. Amino acid residues 1 to 94 of the wild-type human HRAS protein and the wild-type human KRAS protein are 100% identical. Hereinafter, references to "RAS" (mutated or non-mutated (WT)) refer collectively to KRAS, HRAS, and NRAS unless otherwise specified.

[0019] In an embodiment of the present invention, the mutant human RAS amino acid sequence comprises a WT RAS amino acid sequence in which a glycine at position 12 is substituted, where position 12 is defined with reference to the WT RAS protein. The WT RAS protein may be any of the WT KRAS protein (SEQ ID NO: 9 or 10), the WT HRAS protein (SEQ ID NO: 11), or the WT NRAS protein (SEQ ID NO: 12), because, as explained above, amino acid residues 1-86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical, and amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are also 100% identical. Therefore, the amino acid residue at position 12 of the WT KRAS, WT HRAS, and WT NRAS proteins is the same, i.e., glycine.

[0020] The glycine at position 12 of the WT RAS amino acid sequence can be substituted with any amino acid residue other than glycine. In embodiments of the invention, the substitution is a substitution of valine for the glycine at position 12 of the WT RAS amino acid sequence. In this regard, embodiments of the invention provide TCRs with antigen specificity for any WT RAS protein, polypeptide, or peptide amino acid sequence having a G12V mutation.

[0021] RAS mutations and substitutions are defined herein with reference to the amino acid sequence of the WT RAS protein. Accordingly, RAS mutations and substitutions are described herein with reference to the amino acid residue present at a particular position in the WT RAS protein, followed by the position number, followed by the amino acid residue that replaces that residue in the particular mutation or substitution under consideration. A RAS amino acid sequence (e.g., a RAS peptide) may contain fewer than all of the amino acid residues in a full-length WT RAS protein. Accordingly, position 12 is defined herein with reference to the WT full-length RAS protein (i.e., any one of SEQ ID NOS: 9-12), with the understanding that the actual positions of the corresponding residues may differ in particular instances of RAS amino acid sequences. When a position is as defined by any one of SEQ ID NOS: 9-12, the term "G12" refers to the glycine normally present at position 12 in any one of SEQ ID NOS: 9-12, and "G12V" indicates that the glycine normally present at position 12 in any one of SEQ ID NOS: 9-12 has been replaced with a valine. For example, a specific example of a RAS amino acid sequence is, e.g., TEYKLVVVGA G GVGKSALTIQLI (SEQ ID NO: 28) (an exemplary WT KRAS peptide corresponding to consecutive amino acid residues 2-24 of SEQ ID NO: 9), "G12V" refers to the substitution of the underlined glycine in SEQ ID NO: 28 with valine, even if the actual position of the underlined glycine in SEQ ID NO: 28 is 11. Hereinafter, a human RAS amino acid sequence having a G12V mutation is referred to as "G12V RAS."

[0022] Examples of full-length RAS proteins with the G12V mutation are listed in Table 1 below.

[0023] [Table 1]

[0024] In an embodiment of the present invention, a TCR has antigen specificity for a RAS peptide having the G12V mutation, and the mutant RAS peptide has any length. In an embodiment of the present invention, the mutant RAS peptide has any length suitable for binding to any of the HLA class II molecules described herein. For example, a TCR may have antigen specificity for a RAS peptide having a G12V mutation, the RAS peptide having a length of approximately 24 amino acid residues. The mutant RAS peptide may include any consecutive amino acid residues of a mutant RAS protein containing a G12V mutation. In an embodiment of the present invention, a TCR may have antigen specificity for a RAS peptide having a G12V mutation, the mutant RAS peptide having a length of approximately 24 amino acid residues. An example of a specific peptide having a G12V mutation that can be recognized by the G12V TCR of the present invention is the 24-mer MTEYKLVVVGAVGVGKSALTIQLI (SEQ ID NO: 30), and SEQ ID NO: 27 is the wild-type version of the peptide. In an embodiment of the present invention, a TCR has antigen specificity for the mutant human RAS amino acid sequence of SEQ ID NO: 30. In an embodiment of the invention, the TCR does not have antigen specificity for the wild-type human RAS amino acid sequence of SEQ ID NO: 27. Without wishing to be bound by theory, the 24-mer of SEQ ID NO: 30 may be processed and exist in smaller segments.

[0025] In an embodiment of the present invention, the TCR of the present invention can recognize mutant RAS presented by HLA class II molecules. In this regard, the TCR can induce an immune response when it binds to mutant RAS within the framework of HLA class II molecules. The TCR of the present invention can bind to HLA class II molecules in addition to mutant RAS.

[0026] In an embodiment of the present invention, the HLA class II molecule is an HLA-DR molecule. The HLA-DR molecule is a heterodimer of an α chain (DPA) and a β chain (DPB). The HLA-DRA chain may be any HLA-DRA chain. The HLA-DRB chain may be any HLA-DRB chain. In an embodiment of the present invention, the HLA class II molecule is a heterodimer of an HLA-DPA1 chain and an HLA-DPB1 chain. Examples of HLA-DPA1 molecules include, but are not limited to, those encoded by the HLA-DPA1*01:03 or 02:02 allele. Examples of HLA-DPB1 molecules include, but are not limited to, those encoded by the HLA-DPB1*03:01 allele. Preferably, the HLA class II molecule is a heterodimer of an HLA-DPA1*01:03 or 02:02 chain and an HLA-DPB1*03:01 chain.

[0027] The TCRs of the present invention may provide any one or more of a variety of advantages, including when expressed by cells used in adoptive cell transfer. Mutant RAS is expressed in cancer cells and not in normal non-cancer cells. Without being bound by a particular theory or mechanism, it is believed that the TCRs of the present invention advantageously target cancer cells for destruction while minimizing or eliminating destruction of normal non-cancer cells, thereby reducing toxicity. Furthermore, the TCRs of the present invention can advantageously successfully treat or prevent mutant RAS-positive cancers that do not respond to other types of treatment, such as chemotherapy, surgery, or radiation. RAS G12Mutations are among the most common hotspot mutations found in many cancer types. For example, the KRAS G12V mutation occurs in approximately 27% and 9% of pancreatic and colorectal cancer patients, respectively. Furthermore, members of the RAS family share G12 hotspot mutations in different cancer types (e.g., NRAS in melanoma). Furthermore, the TCRs of the present invention can provide highly avid recognition of mutant RAS, which may provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, transfected with vectors encoding one or both of mutant RAS and HLA-DRB1*03:01, pulsed with a RAS peptide carrying the G12V mutation, or a combination thereof). Furthermore, the HLA-DPB1*03:01 allele is expressed in approximately 19% of Caucasian individuals in the United States. Thus, the TCRs of the present invention can expand the number of immunotherapy-eligible cancer patients to include patients who express HLA-DPB1*03:01, who may not be eligible for immunotherapy using TCRs that recognize RAS presented by other MHC molecules. Furthermore, the TCRs, polypeptides, and proteins of the present invention comprise human amino acid sequences, which can reduce the risk of rejection by the human immune system compared to, for example, TCRs, polypeptides, and proteins comprising murine amino acid sequences.

[0028] The term "antigen specificity" as used herein means that a TCR can specifically bind to and immunologically recognize mutant RAS with high avidity. For example, a TCR can express about 1 x 10 TCRs when co-cultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of mutant RAS peptide (e.g., about 0.05 ng / mL to about 10 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, or a range defined by any two of the above values) or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the mutant RAS has been introduced so that the target cells express the mutant RAS.4 ~Approx. 1×10 5 T cells may be considered to have "antigen specificity" for a mutant RAS if they secrete IFN-γ at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the above values). Cells expressing the TCR of the present invention can also secrete IFN-γ when co-cultured with antigen-negative, HLA class II molecule-positive target cells pulsed with a higher concentration of the mutant RAS peptide. The HLA class II molecule may be any of the HLA class II molecules described herein (e.g., HLA-DRB1*03:01 molecule).

[0029] Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for a mutant RAS if T cells expressing the TCR secrete at least two-fold more IFN-γ when co-cultured with either (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of the mutant RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the mutant RAS has been introduced such that the target cells express the mutant RAS, compared to the amount of IFN-γ expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR cocultured with (a) the same concentration of an irrelevant peptide (e.g., another peptide having a different sequence from the mutant RAS peptide) or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the irrelevant peptide has been introduced so that the target cells express the irrelevant peptide, or (ii) non-transduced T cells (e.g., derived from PBMCs that do not express the TCR) cocultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with the same concentration of the mutant RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the mutant RAS has been introduced so that the target cells express the mutant RAS. The HLA class II molecule expressed by the negative control target cells is the same HLA class II molecule expressed by the target cells cocultured with the test T cells. The HLA class II molecule can be any of the HLA class II molecules described herein (e.g., an HLA-DRB1*03:01 molecule). IFN-γ secretion can be measured by methods known in the art, for example, enzyme-linked immunosorbent assay (ELISA).

[0030] Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for a mutant RAS if at least two times as many T cells expressing the TCR secrete IFN-γ when cocultured with (a) antigen-negative HLA class II molecule-positive target cells pulsed with a low concentration of a mutant RAS peptide or (b) antigen-negative HLA class II molecule-positive target cells into which a nucleotide sequence encoding the mutant RAS has been introduced such that the target cells express the mutant RAS, compared to the number of negative control T cells secreting IFN-γ. The HLA class II molecule, peptide concentration, and negative control may be as described herein for other aspects of the invention. The number of IFN-γ-secreting cells can be measured by methods known in the art, e.g., ELISPOT.

[0031] Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for a mutant RAS if T cells expressing the TCR upregulate expression of one or more T cell activation markers, as measured, for example, by flow cytometry, following stimulation with target cells expressing the mutant RAS. Examples of T cell activation markers include 4-1BB, OX40, CD107a, CD69, and cytokines that are upregulated upon antigenic stimulation (e.g., tumor necrosis factor (TNF), interleukin (IL)-2, etc.).

[0032] Embodiments of the present invention provide TCRs comprising two polypeptides (i.e., polypeptide chains), such as, for example, a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, or a combination thereof. The polypeptides of the TCRs of the present invention may comprise any amino acid sequence, so long as the TCR has antigen specificity for a mutant RAS. In some embodiments, the TCR is not naturally occurring.

[0033] In an embodiment of the invention, a TCR comprises two polypeptide chains each comprising a variable region comprising TCR complementarity determining region (CDR) 1, CDR2, and CDR3. In an embodiment of the invention, a TCR comprises a first polypeptide chain comprising CDR1 (α chain CDR1) comprising the amino acid sequence of SEQ ID NO: 1, CDR2 (α chain CDR2) comprising the amino acid sequence of SEQ ID NO: 2, and CDR3 (α chain CDR3) comprising the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising CDR1 (β chain CDR1) comprising the amino acid sequence of SEQ ID NO: 4, CDR2 (β chain CDR2) comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 (β chain CDR3) comprising the amino acid sequence of SEQ ID NO: 6.

[0034] In another embodiment of the invention, the TCR comprises a first polypeptide chain comprising CDR1 (CDR1 of the α chain) comprising the amino acid sequence of SEQ ID NO: 31, CDR2 (CDR2 of the α chain) comprising the amino acid sequence of SEQ ID NO: 32, and CDR3 (CDR3 of the α chain) comprising the amino acid sequence of SEQ ID NO: 33, and a second polypeptide chain comprising CDR1 (CDR1 of the β chain) comprising the amino acid sequence of SEQ ID NO: 34, CDR2 (CDR2 of the β chain) comprising the amino acid sequence of SEQ ID NO: 35, and CDR3 (CDR3 of the β chain) comprising the amino acid sequence of SEQ ID NO: 36.

[0035] In this regard, the TCR of the present invention may comprise any one or more of the amino acid sequences selected from SEQ ID NOs: 1 to 6 and 31 to 36. In an embodiment of the present invention, the TCR comprises the amino acid sequences of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 31 to 33, (d) all of SEQ ID NOs: 34 to 36, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 31 to 36. In a particularly preferred embodiment, the TCR comprises the amino acid sequences of (i) all of SEQ ID NOs: 1 to 6 or (ii) all of SEQ ID NOs: 31 to 36.

[0036] The CDR3 of any one or more of SEQ ID NOs: 3, 6, 33, or 36, i.e., the α chain or the β chain or both, may further comprise a cysteine ​​immediately N-terminal to the first amino acid of the CDR, or a phenylalanine immediately C-terminal to the last amino acid, or both.

[0037] In embodiments of the invention, the TCR comprises the amino acid sequence of a variable region of a TCR comprising the above-described CDRs. The TCR may comprise a human variable region, e.g., a human α chain variable region and a human β chain variable region. In this regard, the TCR may comprise a variable region of the 4360TCR1 α chain with a WT N-terminal signal peptide; a variable region of the 4360TCR1 α chain with a variant N-terminal signal peptide; a variable region of the 4360TCR1 β ... SEQ ID NO: 38 (variable region of 4360 TCR5 α chain with variant N-terminal signal peptide); SEQ ID NO: 47 (variable region of 4360 TCR1 α chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 48 (variable region of 4360 TCR1 β chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 49 (variable region of 4360 TCR5 α chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 50 (variable region of 4360 TCR5 β chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 63 (variable region of 4360 TCR1 α chain with variant N-terminal signal peptide); SEQ ID NO: 130 (variable region of 4360 TCR1 α chain with alternative variant N-terminal signal peptide); SEQ ID NO: 64 (WT SEQ ID NO: 67 (variable region of 4360 TCR1 α chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 68 (variable region of 4360 TCR1 β chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 72 (variable region of 4360 TCR5 α chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 73 (variable region of 4360 TCR5 β chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 65 (variable region of 4360 TCR1 β chain with alternative variant N-terminal signal peptide); SEQ ID NO: 66 (variable region of 4360 TCR1 β chain with alternative WT N-terminal signal peptide);SEQ ID NO: 69 (variable region of 4360 TCR5 β chain with alternative variant N-terminal signal peptide); SEQ ID NO: 70 (variable region of 4360 TCR5 β chain WT N-terminal signal peptide); SEQ ID NO: 71 (variable region of 4360 TCR5 β chain with alternative WT N-terminal signal peptide); SEQ ID NO: 76 (alternative variable region of 4360 TCR1 β chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 102 (alternative variable region of 4360 TCR5 β chain without N-terminal signal peptide predicted using SignalP); both SEQ ID NOs: 7 and 8; both SEQ ID NOs: 129 and 8; both SEQ ID NOs: 63 and 8; both SEQ ID NOs: 130 and 8; both SEQ ID NOs: 7 and 64; both SEQ ID NOs: 129 and 64; both SEQ ID NOs: 63 and 64; both SEQ ID NOs: 130 and 64; SEQ ID NO: 7 and 65; both SEQ ID NOs: 129 and 65; both SEQ ID NOs: 63 and 65; both SEQ ID NOs: 130 and 65; both SEQ ID NOs: 7 and 66; both SEQ ID NOs: 129 and 66; both SEQ ID NOs: 63 and 66; both SEQ ID NOs: 130 and 66; both SEQ ID NOs: 37 and 38; both SEQ ID NOs: 37 and 69; both SEQ ID NOs: 37 and 70; both SEQ ID NOs: 37 and 71; both SEQ ID NOs: 47 and 48; both SEQ ID NOs: 67 and 68; both SEQ ID NOs: 67 and 76; both SEQ ID NOs: 49 and 50; both SEQ ID NOs: 72 and 73; or both SEQ ID NOs: 72 and 102. Preferably, the TCR comprises the amino acid sequence of (i) both SEQ ID NOs: 7 and 8; (ii) both SEQ ID NOs: 63 and 64; (iii) both SEQ ID NOs: 7 and 65; (iv) both SEQ ID NOs: 63 and 66; (v) both SEQ ID NOs: 37 and 38; (vi) both SEQ ID NOs: 37 and 70; (vii) both SEQ ID NOs: 47 and 48; (viii) both SEQ ID NOs: 67 and 68; (ix) both SEQ ID NOs: 67 and 76; (x) both SEQ ID NOs: 49 and 50; (xi) both SEQ ID NOs: 72 and 73; or (xii) both SEQ ID NOs: 72 and 102.

[0038] The TCRs of the invention may further comprise an alpha chain constant region and a beta chain constant region. The constant regions may be derived from any suitable species, such as, for example, human or mouse. In embodiments of the invention, the TCR further comprises mouse alpha and beta chain constant regions or human alpha and beta chain constant regions. As used herein, the terms "mouse" or "human," when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining regions (CDRs), variable region, constant region, alpha chain, and / or beta chain), refer to a TCR (or component thereof) of mouse or human origin, respectively, i.e., a TCR (or component thereof) that originates from or was once expressed by a mouse T cell or a human T cell, respectively.

[0039] Embodiments of the present invention provide chimeric TCRs comprising a human variable region and a mouse constant region, the TCR having antigen specificity for a mutant human RAS amino acid sequence presented by an HLA class II molecule. The mouse constant region may provide any one or more advantages. For example, the mouse constant region may reduce mispairing between the TCR of the present invention and an endogenous TCR of a host cell into which the TCR of the present invention is introduced. Alternatively, or in addition, the mouse constant region may increase expression of the TCR of the present invention compared to the same TCR having a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 19 (wild-type (WT) mouse α chain constant region), SEQ ID NO: 20 (WT mouse β chain constant region), SEQ ID NO: 74 (variant mouse α chain constant region), SEQ ID NO: 75 (variant mouse β chain constant region), or both SEQ ID NOs: 19 and 20 or 74 and 75. Preferably, the TCR of the present invention comprises the amino acid sequences of SEQ ID NOs: 19 and 20 or 74 and 75. A chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the CDR regions described herein with respect to other aspects of the invention. In this regard, the TCR may comprise, for example, the amino acid sequences of: (a) all of SEQ ID NOS: 1-3 and 19; (b) all of SEQ ID NOS: 4-6 and 20; (c) all of SEQ ID NOS: 1-3 and 74; (d) all of SEQ ID NOS: 4-6 and 75; (e) all of SEQ ID NOS: 31-33 and 19; (f) all of SEQ ID NOS: 34-36 and 20; (g) all of SEQ ID NOS: 31-33 and 74; (h) all of SEQ ID NOS: 34-36 and 75; (i) all of SEQ ID NOS: 1-6 and 19-20; (j) all of SEQ ID NOS: 1-6 and 74-75; (k) all of SEQ ID NOS: 31-36 and 19-20; or (l) all of SEQ ID NOS: 31-36 and 74-75. In another embodiment of the invention, the chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the variable regions described herein with respect to other aspects of the invention.In this regard, the TCR may comprise, for example, the amino acid sequences of: (i) both SEQ ID NOs: 7 and 19; (ii) both SEQ ID NOs: 129 and 19; (iii) both SEQ ID NOs: 8 and 20; (iv) both SEQ ID NOs: 7 and 74; (v) both SEQ ID NOs: 129 and 74; (vi) both SEQ ID NOs: 8 and 75; (vii) both SEQ ID NOs: 37 and 19; (viii) both SEQ ID NOs: 38 and 20; (ix) both SEQ ID NOs: 37 and 74; (x) both SEQ ID NOs: 38 and 75; (xi) all of SEQ ID NOs: 7-8 and 19-20; (xii) all of SEQ ID NOs: 129, 8, and 19-20; (xiii) all of SEQ ID NOs: 37-38 and 19-20; (xiv) all of SEQ ID NOs: 7-8 and 74-75; (xv) all of SEQ ID NOs: 129, 8, and 74-75; or (xvi) all of SEQ ID NOs: 37-38 and 74-75.

[0040] In another embodiment of the invention, the TCR is selected from the group consisting of SEQ ID NO: 23 (alpha chain of 4360TCR1 with WT mouse constant region and WT N-terminal signal peptide), SEQ ID NO: 133 (alpha chain of 4360TCR1 with WT mouse constant region and another WT N-terminal signal peptide), SEQ ID NO: 24 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 39 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 40 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 41 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 42 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 43 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 44 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 45 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 46 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 47 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 48 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 49 (beta chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ SEQ ID NO: 40 (beta chain of 4360TCR5 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 51 (alpha chain of 4360TCR1 with WT mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 52 (beta chain of 4360TCR1 with WT mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 53 (alpha chain of 4360TCR5 with WT mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 54 (beta chain of 4360TCR5 with WT mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 77 (alpha chain of 4360TCR1 with replaced mouse constant region and WT N-terminal signal peptide), SEQ ID NO: 132 (replaced mouse constant region and another WT SEQ ID NO: 78 (alpha chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 81 (alpha chain of 4360TCR1 with substituted mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 135 (alpha chain of 4360TCR1 with substituted mouse constant region and alternative variant N-terminal signal peptide), SEQ ID NO: 82 (beta chain of 4360TCR1 with substituted mouse constant region and WT N-terminal signal peptide), SEQ ID NO: 83 (alpha chain of 4360TCR1 with WT mouse constant region and variant N-terminal signal peptide), SEQ ID NO: 136 (alpha chain of 4360TCR1 with WT mouse constant region and alternative variant N-terminal signal peptide), SEQ ID NO: 84 (alpha chain of 4360TCR1 with WT mouse constant region and WT4360TCR1 beta chain with an N-terminal signal peptide), SEQ ID NO: 91 (4360TCR1 alpha chain with a replaced mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 92 (4360TCR1 beta chain with a replaced mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 95 (4360TCR1 alpha chain with a replaced mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 96 (4360TCR1 with a replaced mouse constant region and no N-terminal signal peptide as predicted by SignalP) 0TCR1 beta chain), SEQ ID NO: 97 (4360TCR1 alpha chain with WT mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 98 (4360TCR1 beta chain with WT mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 86 (4360TCR1 beta chain with substituted mouse constant region and alternative variant N-terminal signal peptide), SEQ ID NO: 87 (4360TCR1 beta chain with WT mouse constant region and alternative variant N-terminal signal peptide), SEQ ID NO: 89 (4360TCR1 beta chain with substituted mouse constant region and alternative variant N-terminal signal peptide), β chain of 4360TCR1 with an N-terminal signal peptide), SEQ ID NO: 90 (β chain of 4360TCR1 with a WT mouse constant region and an alternative WT N-terminal signal peptide), SEQ ID NO: 100 (another β chain of 4360TCR1 with a substituted mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 101 (another β chain of 4360TCR1 with a WT mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 103 (α chain of 4360TCR5 with a substituted mouse constant region and a WT N-terminal signal peptide), SEQ ID NO: 104 (β chain of 4360TCR5 with a substituted mouse constant region and a variant N-terminal signal peptide), SEQ ID NO: 106 (β chain of 4360TCR5 with a substituted mouse constant region and a WT N-terminal signal peptide), SEQ ID NO: 107 (WT mouse constant region and a WTSEQ ID NO: 108 (the beta chain of 4360TCR5 with a replaced mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 109 (the beta chain of 4360TCR5 with a replaced mouse constant region and no N-terminal signal peptide as predicted by IMGT), SEQ ID NO: 118 (the alpha chain of 4360TCR5 with a replaced mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 119 (the alpha chain of 4360TCR5 with a replaced mouse constant region and no N-terminal signal peptide as predicted by SignalP). 0TCR5 beta chain), SEQ ID NO: 120 (the alpha chain of 4360TCR5 with WT mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 121 (the beta chain of 4360TCR5 with WT mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 111 (the beta chain of 4360TCR5 with a substituted mouse constant region and an alternative variant N-terminal signal peptide), SEQ ID NO: 112 (the beta chain of 4360TCR5 with a WT mouse constant region and an alternative variant N-terminal signal peptide), SEQ ID NO: 114 (the beta chain of 4360TCR5 with a substituted mouse constant region and an alternative WT N-terminal signal peptide), SEQ ID NO: 115 (the beta chain of 4360TCR5 with a WT mouse constant region and an alternative WTSEQ ID NO: 123 (another β chain of 4360TCR5 with a substituted mouse constant region and no N-terminal signal peptide as predicted by SignalP), SEQ ID NO: 124 (another β chain of 4360TCR5 with a WT mouse constant region and no N-terminal signal peptide as predicted by SignalP), both of SEQ ID NOs: 23-24, both of SEQ ID NOs: 133-24, both of SEQ ID NOs: 39-40, sequence Both SEQ ID NOS: 51-52, Both SEQ ID NOS: 53-54, Both SEQ ID NOS: 77-78, Both SEQ ID NOS: 132-78, Both SEQ ID NOS: 81-82, Both SEQ ID NOS: 135-82, Both SEQ ID NOS: 83-84, Both SEQ ID NOS: 136-84, Both SEQ ID NOS: 91-92, Both SEQ ID NOS: 95-96, Both SEQ ID NOS: 97-98, Both SEQ ID NOS: 103-104, Both SEQ ID NOS: 108-109, Both SEQ ID NOS: 118-119, Both SEQ ID NOS: 120-121 , both SEQ ID NOs: 23 and 90, both SEQ ID NOs: 133 and 90, both SEQ ID NOs: 23 and 87, both SEQ ID NOs: 133 and 87, both SEQ ID NOs: 83 and 90, both SEQ ID NOs: 136 and 90, both SEQ ID NOs: 83 and 87, both SEQ ID NOs: 136 and 87, both SEQ ID NOs: 77 and 86, both SEQ ID NOs: 132 and 86, both SEQ ID NOs: 77 and 89, both SEQ ID NOs: 132 and 89, both SEQ ID NOs: 81 and 89, both SEQ ID NOs: 135 and 89, both SEQ ID NOs: 81 and 86, both SEQ ID NOs: 135 and 86, both SEQ ID NOs: 97 and 101, both SEQ ID NOs: 95 and 100, both SEQ ID NOs: 39 and 106, both SEQ ID NOs: 39 and 112, both SEQ ID NOs: 39 and 115, both SEQ ID NOs: 103 and 107, both SEQ ID NOs: 103 and 111, both SEQ ID NOs: 103 and 114, both SEQ ID NOs: 120 and 124, or both SEQ ID NOs: 118 and 123.

[0041] In embodiments of the invention, a TCR comprises an α chain comprising a variable region and a constant region, and a β chain comprising a variable region and a constant region. In this regard, a TCR may comprise, for example, (a) (i) X at position 179 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 243 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO:21 is Gly, (b) an alpha chain comprising the amino acid sequence of SEQ ID NO: 21 (the alpha chain of 4360TCR1 with a wild-type N-terminal signal peptide), wherein X at position 180 of SEQ ID NO: 131 is Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) (i) X at position 180 of SEQ ID NO: 131 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 24 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 131, wherein X at position 6 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 131, wherein X at position 247 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR1 with a wild-type N-terminal signal peptide); (c) an alpha chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 198 is Ser or Cys. a beta chain comprising the amino acid sequence (the beta chain of 4360TCR1 with a variant N-terminal signal peptide); (d) (i) X at position 179 of SEQ ID NO: 41 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 41 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp;and (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 41, wherein X at position 246 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (alpha chain of 4360TCR5 with a WT N-terminal signal peptide); (e) a beta chain comprising the amino acid sequence of SEQ ID NO: 42, wherein X at position 197 of SEQ ID NO: 42 is Ser or Cys (beta chain of 4360TCR5 with a variant N-terminal signal peptide); (f) both (a) and (c); (g) both (b) and (c); (h) both (d) and (e); (i) (i) X at position 160 of SEQ ID NO: 55 is Thr or Cys; (ii) a beta chain comprising the amino acid sequence of SEQ ID NO: 55, wherein X at position 197 of SEQ ID NO: 55 is Ser or Cys; (iii) X at position 226 of SEQ ID NO: 55 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 227 of SEQ ID NO: 55 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. (j) a β chain comprising the amino acid sequence of SEQ ID NO: 56, in which X at position 173 of SEQ ID NO: 56 is Ser or Cys (the β chain of 4360TCR1 ..., Ala, Val, Leu, Ile, Pr, or the like); (k) (i) X at position 159 of SEQ ID NO: 57 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 57 is Ser, Ala, Val, Leu, Ile, Pr, or the like (iii) X at position 225 of SEQ ID NO: 57 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 226 of SEQ ID NO: 57 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR5 without the N-terminal signal peptide as predicted by IMGT);(l) a beta chain comprising the amino acid sequence of SEQ ID NO: 58, wherein X at position 172 of SEQ ID NO: 58 is Ser or Cys (the beta chain of 4360TCR5 without an N-terminal signal peptide as predicted by IMGT); (m) both (i) and (j); (n) both (k) and (l); (o) an alpha chain comprising the amino acid sequence of SEQ ID NO: 79, wherein (i) X at position 179 of SEQ ID NO: 79 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 79 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 79 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 79 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (a beta chain of 4360TCR5 without an N-terminal signal peptide as predicted by IMGT); (p) an α chain comprising the amino acid sequence of SEQ ID NO: 134, wherein (i) X at position 180 of SEQ ID NO: 134 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 134 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 134 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 134 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (α chain of 4360TCR1 with a variant N-terminal signal peptide); (q) a β chain comprising the amino acid sequence of SEQ ID NO: 80, wherein X at position 198 of SEQ ID NO: 80 is Ser or Cys (WT (r) a beta chain comprising the amino acid sequence of SEQ ID NO: 105, wherein X at position 197 of SEQ ID NO: 105 is Ser or Cys (WT beta chain of 4360TCR5 with N-terminal signal peptide); (s) both (o) and (q); (t) both (p) and (q); (u) both (d) and (r); (v) (i) wherein X at position 159 of SEQ ID NO: 93 is Thr or Cys;(ii) X at position 223 of SEQ ID NO: 93 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 93 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 226 of SEQ ID NO: 93 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. (w) a beta chain comprising the amino acid sequence of SEQ ID NO: 94, wherein X at position 177 of SEQ ID NO: 94 is Ser or Cys (a beta chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP); (x) (i) X at position 158 of SEQ ID NO: 116 is Thr or Cys; (ii) X at position 222 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) a beta chain comprising the amino acid sequence of SEQ ID NO: 94, wherein X at position 177 of SEQ ID NO: 94 is Ser or Cys; (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 116, wherein X at position 224 of SEQ ID NO: 116 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 116, wherein X at position 225 of SEQ ID NO: 116 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP); (y) an alpha chain comprising the amino acid sequence of SEQ ID NO: 117, wherein X at position 176 of SEQ ID NO: 117 is Ser or Cys. (z) both (v) and (w); (aa) both (x) and (y); (bb) a β chain comprising the amino acid sequence of SEQ ID NO: 85, wherein X at position 187 of SEQ ID NO: 85 is Ser or Cys (another β chain of 4360TCR1 with a variant N-terminal signal peptide); (cc) a β chain comprising the amino acid sequence of SEQ ID NO: 88, wherein X at position 187 of SEQ ID NO: 88 is Ser or Cys (another β chain of 4360TCR1 with a WT N-terminal signal peptide);(dd) a β chain comprising the amino acid sequence of SEQ ID NO: 99, wherein X at position 172 of SEQ ID NO: 99 is Ser or Cys (another β chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP); (ee) both (a) and (bb); (ff) both (b) and (bb); (gg) both (o) and (cc); (hh) both (p) and (cc); (ii) both (v) and (dd); (jj) a β chain comprising the amino acid sequence of SEQ ID NO: 110, wherein X at position 186 of SEQ ID NO: 110 is Ser or Cys (another β chain of 4360TCR5 with a variant N-terminal signal peptide); (kk) a β chain comprising the amino acid sequence of SEQ ID NO: 113, wherein X at position 186 of SEQ ID NO: 113 is Ser or Cys (WT (ll) a beta chain comprising the amino acid sequence of SEQ ID NO: 122, wherein X at position 171 of SEQ ID NO: 122 is Ser or Cys (another beta chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP); (mm) both (d) and (jj); (nn) both (d) and (kk); or (oo) (x); and (ll). In an embodiment of the invention, a TCR comprising SEQ ID NO: 21 does not comprise SEQ ID NO: 23 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 131 does not comprise SEQ ID NO: 133 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 22 does not comprise SEQ ID NO: 24 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 41 does not comprise SEQ ID NO: 39 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 42 does not comprise SEQ ID NO: 40 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 55 does not comprise SEQ ID NO: 51 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 56 does not comprise SEQ ID NO: 52 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 57 does not comprise SEQ ID NO: 53 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 58 does not comprise SEQ ID NO: 54 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 79 does not comprise SEQ ID NO: 83 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 134 does not comprise SEQ ID NO: 136 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 80 does not comprise SEQ ID NO: 84 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 93 does not comprise SEQ ID NO: 97 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 94 does not comprise SEQ ID NO: 98 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 85 does not comprise SEQ ID NO: 87 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 88 does not comprise SEQ ID NO: 90 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 99 does not comprise SEQ ID NO: 101 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 116 does not comprise SEQ ID NO: 120 (unsubstituted alpha chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 117 does not comprise SEQ ID NO: 121 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 105 does not comprise SEQ ID NO: 107 (unsubstituted beta chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 110 does not comprise SEQ ID NO: 112 (unsubstituted β chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 113 does not comprise SEQ ID NO: 115 (unsubstituted β chain).In an embodiment of the invention, a TCR comprising SEQ ID NO: 122 does not comprise SEQ ID NO: 124 (unsubstituted β chain).

[0042] The first amino acid of any of the mouse alpha constant regions described herein can be different from N as provided in SEQ ID NOs: 17 and 19. For example, in any TCR construct, polypeptide, protein, etc. described herein, this first amino acid can be encoded by a split codon (with nucleotides from both the variable and constant regions), such that any of the mouse alpha constant regions can have a different amino acid at that position. Similarly, the first amino acid of any of the mouse beta constant regions described herein can be different from E as provided in SEQ ID NOs: 18 and 20, e.g., this first amino acid can be encoded by a split codon.

[0043] In embodiments of the invention, the TCR comprises a substituted constant region. In this regard, the TCR may comprise the amino acid sequence of any of the TCRs described herein, for example, with one, two, three, or four amino acid substitution(s) in the constant regions of one or both of the α and β chains. Preferably, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in the murine constant region of one or both of the α and β chains. In particularly preferred embodiments, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in the murine constant region of the α chain and one amino acid substitution in the murine constant region of the β chain. In some embodiments, TCRs comprising a substituted constant region advantageously exhibit a mutant RAS activity compared to a parent TCR comprising an unsubstituted (wild-type) constant region. +These provide one or more of: increased target recognition, increased expression by host cells, decreased mispairing with endogenous TCRs, and increased anti-tumor activity. Generally, the substituted amino acid sequences of the murine constant regions of the TCR α and β chains, SEQ ID NOs: 17 and 18, respectively, correspond to all or part of the amino acid sequences of the unsubstituted murine constant regions, SEQ ID NOs: 19 and 20, respectively, with SEQ ID NO: 17 having one, two, three, or four amino acid substitution(s) compared to SEQ ID NO: 19, and SEQ ID NO: 18 having one amino acid substitution compared to SEQ ID NO: 20. In this regard, embodiments of the present invention provide TCRs comprising the amino acid sequences of (a) SEQ ID NO: 17 (α chain constant region), in which (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 is GIy, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) SEQ ID NO: 18 (β chain constant region), in which X at position 57 is Ser or Cys; or (c) both SEQ ID NOs: 17 and 18. In embodiments of the present invention, a TCR comprising SEQ ID NO: 17 does not comprise SEQ ID NO: 19 (unsubstituted murine constant region of the α chain). In an embodiment of the invention, a TCR comprising SEQ ID NO: 18 does not comprise SEQ ID NO: 20 (unsubstituted murine constant region of the β chain).

[0044] In embodiments of the invention, the replaced constant region comprises a cysteine ​​substitution in the constant region of one or both of the α and β chains to provide a cysteine-substituted TCR. Opposing cysteines in the α and β chains provide a disulfide bond that links the α and β chain constant regions of the replaced TCR together and is not present in TCRs comprising unsubstituted mouse constant regions. In this regard, the TCR may be a cysteine-substituted TCR in which one or both of the native Thr at position 48 of SEQ ID NO: 19 (Thr48) and the native Ser at position 57 of SEQ ID NO: 20 (Ser57) may be replaced with Cys. Preferably, both the native Thr at position 48 of SEQ ID NO: 19 and the native Ser at position 57 of SEQ ID NO: 20 are replaced with Cys. Exemplary sequences of the constant regions of cysteine-substituted TCRs are listed in Table 2. In embodiments of the invention, the cysteine ​​replaced TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NOs: 17 and 18, both of which are as defined in Table 2. The cysteine ​​replaced TCRs of the invention may comprise a replaced constant region in addition to any of the CDRs or variable regions described herein.

[0045] In embodiments of the invention, the cysteine-substituted chimeric TCR comprises a full-length alpha chain and a full-length beta chain. Exemplary sequences of the alpha and beta chains of cysteine-substituted chimeric TCRs are set forth in Table 2. In embodiments of the invention, the TCR is selected from the group consisting of: (i) SEQ ID NO:21, (ii) SEQ ID NO:131, (iii) SEQ ID NO:22, (iv) SEQ ID NO:41, (v) SEQ ID NO:42, (vi) both SEQ ID NOs:21 and 22, (vii) both SEQ ID NOs:131 and 22, (viii) both SEQ ID NOs:41 and 42, (ix) SEQ ID NO:55, (x) SEQ ID NO:56, (xi) SEQ ID NO:57, (xii) SEQ ID NO:58, (xiii) both SEQ ID NOs:55 and 56, or (xiv) both SEQ ID NOs: 57 and 58, (xv) SEQ ID NO: 79, (xvi) SEQ ID NO: 134, (xvii) SEQ ID NO: 80, (xviii) SEQ ID NO: 105, (xix) both SEQ ID NOs: 79 and 80, (xx) both SEQ ID NOs: 134 and 80, (xxi) both SEQ ID NOs: 41 and 105, (xxii) SEQ ID NO: 93, (xxiii) SEQ ID NO: 94, (xxiv) SEQ ID NO: 116, (xxv) SEQ ID NO: 117, (xxvi) both SEQ ID NOs: 93 and 94, (xxvii) both SEQ ID NOs: 116 and 117, (xxviii) SEQ ID NO: 85, (xxix) SEQ ID NO: 88, (xxx) SEQ ID NO: 99, (xxxi) both SEQ ID NOs: 21 and 85, (xxxii) both SEQ ID NOs: 131 and 85, (xxxiii) both SEQ ID NOs: 79 and 88, (xxxiv) both SEQ ID NOs: 134 and 88, (xxxv) both SEQ ID NOs: 93 and 99, (xxxvi) SEQ ID NO: 110, (xx xvii) SEQ ID NO: 113, (xxxviii) SEQ ID NO: 122, (xxxix) both SEQ ID NOs: 41 and 110, (xl) both SEQ ID NOs: 41 and 113, (xli) both SEQ ID NOs: 116 and 122, and SEQ ID NOs: 17, 18, 21, 22, 41, 42, 55-58, 79, 80, 85, 88, 93, 94, 99, 105, 110, 113, 116, 117, 122, 131, and 134 are all as defined in Table 2.

[0046] [Table 2-1]

[0047] [Table 2-2]

[0048] [Table 2-3]

[0049] [Table 2-4]

[0050] In embodiments of the invention, the replacement amino acid sequence comprises the substitution of one, two, or three amino acids in the transmembrane (TM) domain of the α chain constant region with hydrophobic amino acids to provide a hydrophobic amino acid-substituted TCR (also referred to herein as an "LVL-modified TCR"). The hydrophobic amino acid substitution(s) in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR compared to a TCR that does not have a hydrophobic amino acid substitution(s) in the TM domain. In this regard, the TCR is an LVL-modified TCR in which one, two, or three of native Ser112, Met114, and Gly115 of SEQ ID NO: 19 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val; and native Ser57 of SEQ ID NO: 20 may be substituted with Cys. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 19 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In embodiments of the invention, the LVL-modified TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NOs: 17 and 18, both of which are as defined in Table 3. The LVL-modified TCRs of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0051] In embodiments of the invention, the LVL-modified TCR comprises a full-length alpha chain and a full-length beta chain. Exemplary sequences of the alpha and beta chains of LVL-modified TCRs are set forth in Table 3. In embodiments of the invention, the LVL-modified TCR comprises (i) SEQ ID NO: 21, (ii) SEQ ID NO: 131, (iii) SEQ ID NO: 22, (iv) SEQ ID NO: 41, (v) SEQ ID NO: 42, (vi) both SEQ ID NOs: 21 and 22, (vii) both SEQ ID NOs: 131 and 22, (viii) both SEQ ID NOs: 41 and 42, (ix) SEQ ID NO: 55, (x) SEQ ID NO: 56, (xi) SEQ ID NO: 57, (xii) SEQ ID NO: 58, (xiii) both SEQ ID NOs: 55 and 56 or (xiv) both SEQ ID NOs: 57 and 58, (xv) SEQ ID NO: 79, (xvi) SEQ ID NO: 134, (xvii) SEQ ID NO: 80, (xviii) SEQ ID NO: 105, (xix) both SEQ ID NOs: 79 and 80, (xx) both SEQ ID NOs: 134 and 80, (xxi) both SEQ ID NOs: 41 and 105, (xxii) SEQ ID NO: 93, (xxiii) SEQ ID NO: 94, (xxiv) SEQ ID NO: 116, (xxv) SEQ ID NO: 117, (xxvi) SEQ ID NO: (xxvii) both SEQ ID NOs: 116 and 117, (xxviii) SEQ ID NO: 85, (xxix) SEQ ID NO: 88, (xxx) SEQ ID NO: 99, (xxxi) both SEQ ID NOs: 21 and 85, (xxxii) both SEQ ID NOs: 131 and 85, (xxxiii) both SEQ ID NOs: 79 and 88, (xxxiv) both SEQ ID NOs: 134 and 88, (xxxv) both SEQ ID NOs: 93 and 99, (xxxvi) SEQ ID NO: 110, (xxxvii) SEQ ID NO: 113, (xxxviii) SEQ ID NO: 122, (xxxix) both SEQ ID NOs: 41 and 110, (xl) both SEQ ID NOs: 41 and 113, (xli) both SEQ ID NOs: 116 and 122, and SEQ ID NOs: 17, 18, 21, 22, 41, 42, 55-58, 79, 80, 85, 88, 93, 94, 99, 105, 110, 113, 116, 117, 122, 131, and 134 are all as defined in Table 3.

[0052] [Table 3-1]

[0053]

Table 3-2

[0054]

Table 3-3

[0055]

Table 3-4

[0056]

Table 3-5

[0057]

Table 3-6

[0058]

Table 3-7

[0059]

Table 3-8

[0060]

Table 3-9

[0061] In embodiments of the invention, the replaced amino acid sequence comprises a cysteine ​​substitution in the constant region of one or both of the α and β chains in combination with substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of the α chain with a hydrophobic amino acid (also referred to herein as a "cysteine-substituted LVL-modified TCR"). In this regard, the TCR is a cysteine-substituted LVL-modified chimeric TCR in which native Thr48 of SEQ ID NO: 19 has been replaced with Cys; one, two, or three of native Ser112, Met114, and Gly115 of SEQ ID NO: 19 have been replaced independently with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val; and native Ser57 of SEQ ID NO: 20 has been replaced with Cys. Preferably, all three of the native Ser112, Met114, and Gly115 of SEQ ID NO: 19 may be independently substituted with Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; preferably Leu, Ile, or Val. In embodiments of the invention, the cysteine-substituted LVL-modified TCR comprises (i) SEQ ID NO: 17, (ii) SEQ ID NO: 18, or (iii) both SEQ ID NOs: 17 and 18, both of which are as defined in Table 4. The cysteine-substituted LVL-modified TCRs of the invention may comprise a substituted constant region in addition to any of the CDRs or variable regions described herein.

[0062] In embodiments, the cysteine-substituted LVL-modified TCR comprises a full-length alpha chain and a full-length beta chain. In embodiments of the invention, the cysteine-substituted LVL-modified TCR is selected from the group consisting of (i) SEQ ID NO:21, (ii) SEQ ID NO:131, (iii) SEQ ID NO:22, (iv) SEQ ID NO:41, (v) SEQ ID NO:42, (vi) both SEQ ID NOs:21 and 22, (vii) both SEQ ID NOs:131 and 22, (viii) both SEQ ID NOs:41 and 42, (ix) SEQ ID NO:55, (x) SEQ ID NO:56, (xi) SEQ ID NO:57, (xii) SEQ ID NO:58, (xiii) SEQ ID NO:55 and and 56, or (xiv) both SEQ ID NOs: 57 and 58, (xv) SEQ ID NO: 79, (xvi) SEQ ID NO: 134, (xvii) SEQ ID NO: 80, (xviii) SEQ ID NO: 105, (xix) both SEQ ID NOs: 79 and 80, (xx) both SEQ ID NOs: 134 and 80, (xxi) both SEQ ID NOs: 41 and 105, (xxii) SEQ ID NO: 93, (xxiii) SEQ ID NO: 94, (xxiv) SEQ ID NO: 116, (xxv) SEQ ID NO: 117, (xxvi) (xxvii) both SEQ ID NOs: 116 and 117, (xxviii) SEQ ID NO: 85, (xxix) SEQ ID NO: 88, (xxx) SEQ ID NO: 99, (xxxi) both SEQ ID NOs: 21 and 85, (xxxii) both SEQ ID NOs: 131 and 85, (xxxiii) both SEQ ID NOs: 79 and 88, (xxxiv) both SEQ ID NOs: 134 and 88, (xxxv) both SEQ ID NOs: 93 and 99, (xxxvi) SEQ ID NO: 110, (xxxvii) SEQ ID NO: 113, (xxxviii) SEQ ID NO: 122, (xxxix) both SEQ ID NOs: 41 and 110, (xl) both SEQ ID NOs: 41 and 113, (xli) both SEQ ID NOs: 116 and 122, and SEQ ID NOs: 17, 18, 21, 22, 41, 42, 55-58, 79, 80, 85, 88, 93, 94, 99, 105, 110, 113, 116, 117, 122, 131, and 134 are all as defined in Table 4.

[0063] [Table 4-1]

[0064] [Table 4-2]

[0065]

Table 4-3

[0066]

Table 4-4

[0067]

Table 4-5

[0068]

Table 4-6

[0069]

Table 4-7

[0070]

Table 4-8

[0071]

Table 4-9

[0072] In an embodiment of the invention, the cysteine-substituted LVL-modified TCR comprises: (a) SEQ ID NO: 74 (α-chain constant region of cysteine-substituted LVL-modified TCR); (b) SEQ ID NO: 75 (β-chain constant region of cysteine-substituted LVL-modified TCR); (c) SEQ ID NO: 77 (WT (d) SEQ ID NO: 78 (β chain of cysteine-substituted LVL-modified 4360TCR1 with a variant N-terminal signal sequence); (e) SEQ ID NO: 91 (α chain of cysteine-substituted LVL-modified 4360TCR1 without the N-terminal signal sequence predicted by IMGT); (f) SEQ ID NO: 92 (β chain of cysteine-substituted LVL-modified 4360TCR1 without the N-terminal signal sequence predicted by IMGT); (g) SEQ ID NO: 95 (α chain of cysteine-substituted LVL-modified 4360TCR1 without the N-terminal signal sequence predicted by SignalP); (h) SEQ ID NO: 96 (β chain of cysteine-substituted LVL-modified 4360TCR1 without the N-terminal signal sequence predicted by SignalP); (i) SEQ ID NO: 81 (α chain of cysteine-substituted LVL-modified 4360TCR1 with a variant N-terminal signal sequence); (j) SEQ ID NO: 82 (WT (k) SEQ ID NO: 89 (another β chain of cysteine-substituted LVL-modified 4360TCR1 with an N-terminal signal sequence); (l) SEQ ID NO: 86 (a β chain of cysteine-substituted LVL-modified 4360TCR1 with a variant N-terminal signal sequence); (m) SEQ ID NO: 100 (a β chain of cysteine-substituted LVL-modified 4360TCR1 without the N-terminal signal sequence predicted by SignalP); (n) SEQ ID NO: 132 (another WT (o) SEQ ID NO: 135 (cysteine-substituted LVL-modified 4360TCR1 alpha chain with an N-terminal signal sequence); (p) both (a) and (b); (q) both (c) and (d); (r) both (e) and (f); (s) both (g) and (h); (t) both (i) and (j); (u) both (i) and (k); (v) both (c) and (l); (w) both (g) and (m); (x) both (n) and (d); or both (o) and (j).

[0073] In an embodiment of the invention, the cysteine-substituted LVL-modified TCR comprises: (a) SEQ ID NO: 74 (α-chain constant region of cysteine-substituted LVL-modified TCR); (b) SEQ ID NO: 75 (β-chain constant region of cysteine-substituted LVL-modified TCR); (c) SEQ ID NO: 103 (WT (d) SEQ ID NO: 104 (β chain of cysteine-substituted LVL-modified 4360TCR5 with a variant N-terminal signal sequence); (e) SEQ ID NO: 108 (α chain of cysteine-substituted LVL-modified 4360TCR5 without the N-terminal signal sequence predicted by IMGT); (f) SEQ ID NO: 109 (β chain of cysteine-substituted LVL-modified 4360TCR5 without the N-terminal signal sequence predicted by IMGT); (g) SEQ ID NO: 118 (α chain of cysteine-substituted LVL-modified 4360TCR5 without the N-terminal signal sequence predicted by SignalP); (h) SEQ ID NO: 119 (β chain of cysteine-substituted LVL-modified 4360TCR5 without the N-terminal signal sequence predicted by SignalP); (j) SEQ ID NO: 106 (WT (k) SEQ ID NO: 114 (another β chain of a cysteine-substituted LVL-modified 4360TCR5 with a WT N-terminal signal sequence); (l) SEQ ID NO: 111 (another β chain of a cysteine-substituted LVL-modified 4360TCR5 with a variant N-terminal signal sequence); (m) SEQ ID NO: 123 (another β chain of a cysteine-substituted LVL-modified 4360TCR5 without the N-terminal signal sequence predicted by SignalP); (n) both (a) and (b); (o) both (c) and (d); (p) both (e) and (f); (q) both (g) and (h); (r) both (c) and (j); (s) both (c) and (k); (t) both (c) and (l); or (u) both (g) and (m).

[0074] Also provided by embodiments of the invention are polypeptides comprising a functional portion of any of the TCRs described herein. The term "polypeptide," as used herein, includes oligopeptides and refers to a single chain of amino acids joined by one or more peptide bonds.

[0075] With respect to the polypeptides of the present invention, a functional portion can be any portion comprising consecutive amino acids of the TCR of which it is a part, so long as it specifically binds to mutant RAS. The term "functional portion," when used in reference to a TCR, refers to any portion or fragment of a TCR of the present invention that retains the biological activity of the TCR of which it is a part (the parent TCR). A functional portion includes, for example, a portion of a TCR that retains the ability to specifically bind to mutant RAS or detect, treat, or prevent cancer (e.g., within the context of an HLA-DPB1*03:01 molecule) to a similar, identical, or greater extent than the parent TCR. With respect to the parent TCR, a functional portion can constitute, for example, about 10%, about 25%, about 30%, about 50%, about 70%, about 80%, about 90%, about 95%, or more of the parent TCR.

[0076] A functional portion may include additional amino acids at the amino or carboxy terminus of the portion, or at both termini, that are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specific binding to mutant RAS; and / or having the ability to detect, treat, or prevent cancer. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0077] The polypeptide may comprise a functional portion of either or both of the α chain and β chain of a TCR of the present invention, for example a functional portion comprising one or more of CDR1, CDR2, and CDR3 of the variable region(s) of the α chain and / or β chain of a TCR of the present invention. In an embodiment of the present invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 (CDR1 of the α chain), SEQ ID NO: 2 (CDR2 of the α chain), SEQ ID NO: 3 (CDR3 of the α chain), SEQ ID NO: 4 (CDR1 of the β chain), SEQ ID NO: 5 (CDR2 of the β chain), SEQ ID NO: 6 (CDR3 of the β chain), or a combination thereof. In another embodiment of the present invention, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 31 (CDR1 of the α chain), SEQ ID NO: 32 (CDR2 of the α chain), SEQ ID NO: 33 (CDR3 of the α chain), SEQ ID NO: 34 (CDR1 of the β chain), SEQ ID NO: 35 (CDR2 of the β chain), SEQ ID NO: 36 (CDR3 of the β chain), or a combination thereof.

[0078] In this regard, the polypeptides of the present invention may comprise any one or more of the amino acid sequences selected from SEQ ID NOs: 1 to 6 and 31 to 36. In embodiments of the present invention, the TCR comprises the amino acid sequence of (a) all of SEQ ID NOs: 1 to 3, (b) all of SEQ ID NOs: 4 to 6, (c) all of SEQ ID NOs: 31 to 33, (d) all of SEQ ID NOs: 34 to 36, (e) all of SEQ ID NOs: 1 to 6, or (f) all of SEQ ID NOs: 31 to 36. In preferred embodiments, the polypeptide comprises the amino acid sequence of (i) all of SEQ ID NOs: 1 to 6 or (ii) all of SEQ ID NOs: 31 to 36. The CDR3 of any one or more of SEQ ID NOs: 3, 6, 33, or 36, i.e., the α chain or the β chain, or both, may further comprise a cysteine ​​immediately N-terminal to the first amino acid of the CDR, or a phenylalanine immediately C-terminal to the last amino acid, or both.

[0079] In embodiments of the invention, polypeptides of the invention may comprise variable regions of TCRs of the invention, for example, comprising a combination of the above CDR regions. In this regard, the TCRs may be selected from SEQ ID NO: 7 (variable region of the 4360TCR1 α chain with a WT N-terminal signal peptide); SEQ ID NO: 129 (variable region of the 4360TCR1 α chain with an alternative WT N-terminal signal peptide); SEQ ID NO: 8 (variable region of the 4360TCR1 β chain with a variant N-terminal signal peptide); SEQ ID NO: 37 (WT SEQ ID NO: 38 (variable region of 4360 TCR5 α chain with variant N-terminal signal peptide); SEQ ID NO: 47 (variable region of 4360 TCR1 α chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 48 (variable region of 4360 TCR1 β chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 49 (variable region of 4360 TCR5 α chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 50 (variable region of 4360 TCR5 β chain without N-terminal signal peptide predicted using IMGT); SEQ ID NO: 63 (variable region of 4360 TCR1 α chain with variant N-terminal signal peptide); SEQ ID NO: 130 (variable region of 4360 TCR1 α chain with alternative variant N-terminal signal peptide); SEQ ID NO: 64 (WT SEQ ID NO: 67 (variable region of 4360TCR1 α chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 68 (variable region of 4360TCR1 β chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 72 (variable region of 4360TCR5 α chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 73 (variable region of 4360TCR5 β chain without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 65 (variable region of 4360TCR1 β chain with alternative variant N-terminal signal peptide); SEQ ID NO: 66 (variable region of 4360TCR1 β chain with alternative WT N-terminal signal peptide); SEQ ID NO: 69 (variable region of 4360TCR5 β chain with alternative variant N-terminal signal peptide);SEQ ID NO: 70 (4360 TCR5 β chain variable region WT N-terminal signal peptide); SEQ ID NO: 71 (4360 TCR5 β chain variable region with alternative WT N-terminal signal peptide); SEQ ID NO: 76 (4360 TCR1 β chain alternative variable region without N-terminal signal peptide predicted using SignalP); SEQ ID NO: 102 (4360 TCR5 β chain alternative variable region without N-terminal signal peptide predicted using SignalP); both SEQ ID NOs: 7 and 8; both SEQ ID NOs: 129 and 8; both SEQ ID NOs: 63 and 8; both SEQ ID NOs: 130 and 8; both SEQ ID NOs: 7 and 64; both SEQ ID NOs: 129 and 64; both SEQ ID NOs: 63 and 64; both SEQ ID NOs: 130 and 64; SEQ ID NO: 7 and 65; both SEQ ID NOs: 129 and 65; both SEQ ID NOs: 63 and 65; both SEQ ID NOs: 130 and 65; both SEQ ID NOs: 7 and 66; both SEQ ID NOs: 129 and 66; both SEQ ID NOs: 63 and 66; both SEQ ID NOs: 130 and 66; both SEQ ID NOs: 37 and 38; both SEQ ID NOs: 37 and 69; both SEQ ID NOs: 37 and 70; both SEQ ID NOs: 37 and 71; both SEQ ID NOs: 47 and 48; both SEQ ID NOs: 67 and 68; both SEQ ID NOs: 67 and 76; both SEQ ID NOs: 49 and 50; both SEQ ID NOs: 72 and 73; or both SEQ ID NOs: 72 and 102. Preferably, the TCR comprises the amino acid sequence of (i) both SEQ ID NOs: 7 and 8; (ii) both SEQ ID NOs: 63 and 64; (iii) both SEQ ID NOs: 7 and 65; (iv) both SEQ ID NOs: 63 and 66; (v) both SEQ ID NOs: 37 and 38; (vi) both SEQ ID NOs: 37 and 70; (vii) both SEQ ID NOs: 47 and 48; (viii) both SEQ ID NOs: 67 and 68; (ix) both SEQ ID NOs: 67 and 76; (x) both SEQ ID NOs: 49 and 50; (xi) both SEQ ID NOs: 72 and 73; or (xii) both SEQ ID NOs: 72 and 102.

[0080] In embodiments of the invention, the polypeptide of the invention may further comprise the constant region of a TCR of the invention as described above. In this regard, the polypeptide may further comprise the amino acid sequence of SEQ ID NO: 19 (WT mouse constant region of the α chain), SEQ ID NO: 20 (WT mouse constant region of the β chain), SEQ ID NO: 17 (substituted mouse constant region of the α chain), SEQ ID NO: 18 (substituted mouse constant region of the β chain), SEQ ID NO: 74 (variant mouse α chain constant region), SEQ ID NO: 75 (variant mouse β chain constant region), both SEQ ID NOs: 19 and 20, both SEQ ID NOs: 17 and 18, or both SEQ ID NOs: 74 and 75. Preferably, the polypeptide further comprises the amino acid sequences of both SEQ ID NOs: 19 and 20, both SEQ ID NOs: 17 and 18, or both SEQ ID NOs: 74 and 75, in combination with any of the CDR regions or variable regions described herein with respect to other aspects of the invention.

[0081] In embodiments of the invention, a polypeptide comprises (a) the amino acid sequence of SEQ ID NO: 17, wherein (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) both (a) and (b). In embodiments of the invention, one or both of SEQ ID NOs: 17 and 18 of the polypeptide are as defined in any one of Tables 2-4. The alpha chain constant regions provided herein are shown with an N-terminal asparagine. In some embodiments, the N-terminal amino acid of the alpha chain constant regions described herein is aspartic acid.

[0082] In an embodiment of the invention, the polypeptide of the invention may comprise the full-length α or β chain of a TCR as described herein. In this regard, polypeptides of the present invention include SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:131; SEQ ID NO:132; SEQ ID NO:133; SEQ ID NO:134; SEQ ID NO:135; SEQ ID NO:136; both SEQ ID NOs:21 and 22, both SEQ ID NOs:131 and 22, both SEQ ID NOs:23 and 24, both SEQ ID NOs:133 and 24, both SEQ ID NOs:77 and 78, both SEQ ID NOs:132 and 78, both SEQ ID NOs:79 and 80, both SEQ ID NOs:134 and 80, both SEQ ID NOs:81 and 82, both SEQ ID NOs:135 and 82, both SEQ ID NOs:83 and 84, both SEQ ID NOs:136 and 84, both SEQ ID NOs:21 and 85, SEQ ID NOs:131 and 85 and 96, both SEQ ID NOs:23 and 87, both SEQ ID NOs:133 and 87, both SEQ ID NOs:77 and 86, both SEQ ID NOs:132 and 86, both SEQ ID NOs:79 and 88, both SEQ ID NOs:134 and 88, both SEQ ID NOs:83 and 90, both SEQ ID NOs:136 and 90, both SEQ ID NOs:81 and 89, SEQ ID NOs:135 and 89, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, both SEQ ID NOs:55 and 56, both SEQ ID NOs:51 and 52, both SEQ ID NOs:91 and 92, both SEQ ID NOs:93 and 94, both SEQ ID NOs:95 and 96, both SEQ ID NOs:97 and 98, both SEQ ID NOs:93 and 99, SEQ ID NOs:95 and 100, or both SEQ ID NOs:97 and 101.In this regard, polypeptides of the present invention include SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, both SEQ ID NOs:41 and 42, both SEQ ID NOs:39 and 40, both SEQ ID NOs:103 and 104, both SEQ ID NOs:41 and 105, both SEQ ID NOs:103 and 106, both SEQ ID NOs:39 and 107, both SEQ ID NOs:41 and 110, both SEQ ID NOs:39 and 112, both SEQ ID NOs:103 and 111, both SEQ ID NOs:41 and 113, SEQ ID NOs:39 and and 115, SEQ ID NO: 103 and 114, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, both SEQ ID NOs: 57 and 58, or both SEQ ID NOs: 53 and 54, both SEQ ID NOs: 108 and 109, both SEQ ID NOs: 116 and 117, both SEQ ID NOs: 118 and 119, both SEQ ID NOs: 120 and 121, both SEQ ID NOs: 116 and 122, both SEQ ID NOs: 120 and 124, or both SEQ ID NOs: 118 and 123. Alternatively, a polypeptide of the invention may comprise both chains of a TCR as described herein.

[0083] In an embodiment of the invention, the polypeptide comprises (a) (i) X at position 179 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 243 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO:21 is GIy, Ala, Val, Leu, Ile, Pro, an alpha chain comprising the amino acid sequence of SEQ ID NO: 21 (the alpha chain of 4360TCR1 with a wild-type N-terminal signal peptide), wherein X is Phe, Met, or Trp; (b) (i) X at position 180 of SEQ ID NO: 131 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 131 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Tr and (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 131, wherein X at position 247 of SEQ ID NO: 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR1 with a wild-type N-terminal signal peptide); (c) a beta chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 198 of SEQ ID NO: 22 is Ser or Cys (the beta chain of 4360TCR1 with a variant N-terminal signal peptide); (d)(i) an amino acid sequence of SEQ ID NO: 41, wherein X at position 179 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR1 with a wild-type N-terminal signal peptide); an alpha chain comprising the amino acid sequence of SEQ ID NO: 41, wherein X is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 41 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR5 with a WT N-terminal signal peptide);(e) a beta chain comprising the amino acid sequence of SEQ ID NO: 42, wherein X at position 197 of SEQ ID NO: 42 is Ser or Cys (beta chain of 4360TCR5 with a variant N-terminal signal peptide); (f) both (a) and (c); (g) both (b) and (c); or (h) both (d) and (e); (i) (i) X at position 160 of SEQ ID NO: 55 is Thr or Cys; (ii) X at position 224 of SEQ ID NO: 55 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) a beta chain comprising the amino acid sequence of SEQ ID NO: 42, wherein X at position 197 of SEQ ID NO: 42 is Ser or Cys; (iv) an α chain comprising the amino acid sequence of SEQ ID NO: 55, wherein X at position 226 of SEQ ID NO: 55 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) an α chain comprising the amino acid sequence of SEQ ID NO: 55, wherein X at position 227 of SEQ ID NO: 55 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR1 without an N-terminal signal peptide as predicted by IMGT); (j) a β chain comprising the amino acid sequence of SEQ ID NO: 56, wherein X at position 173 of SEQ ID NO: 56 is Ser or Cys (the α chain of 4360TCR1 without an N-terminal signal peptide as predicted by IMGT). (k) (i) X at position 159 of SEQ ID NO: 57 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 57 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 57 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 226 of SEQ ID NO: 57 is Gly, Ala, Val, Leu, Ile, Pr (i) an alpha chain comprising the amino acid sequence of SEQ ID NO: 57, wherein X at position 172 of SEQ ID NO: 58 is Ser or Cys (the alpha chain of 4360TCR5 without an N-terminal signal peptide as predicted by IMGT); (l) a beta chain comprising the amino acid sequence of SEQ ID NO: 58, wherein X at position 172 of SEQ ID NO: 58 is Ser or Cys (the beta chain of 4360TCR5 without an N-terminal signal peptide as predicted by IMGT); (m) both (i) and (j); or (n) both (k) and (l); (o)(i) wherein X at position 179 of SEQ ID NO: 79 is Thr or Cys;(ii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 79, wherein X at position 243 of SEQ ID NO: 79 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 79 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 79 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR1 with a variant N-terminal signal peptide); (p) (i) X at position 180 of SEQ ID NO: 134 is Thr or Cys; (iii) an alpha chain comprising the amino acid sequence of SEQ ID NO: 134, wherein X at position 244 of SEQ ID NO: 134 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; and (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 134, wherein X at position 246 of SEQ ID NO: 134 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) an alpha chain comprising the amino acid sequence of SEQ ID NO: 134, wherein X at position 247 of SEQ ID NO: 134 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the alpha chain of 4360TCR1 with a variant N-terminal signal peptide); (q) a beta chain comprising the amino acid sequence of SEQ ID NO: 80, wherein X at position 198 of SEQ ID NO: 80 is Ser or Cys (WT (r) a beta chain comprising the amino acid sequence of SEQ ID NO: 105, wherein X at position 197 of SEQ ID NO: 105 is Ser or Cys (WT beta chain of 4360TCR5 with N-terminal signal peptide); (s) both (o) and (q); (t) both (p) and (q); (u) both (d) and (r); (v) (i) X at position 159 of SEQ ID NO: 93 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 93 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 93 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp;and (iv) an α chain comprising the amino acid sequence of SEQ ID NO: 93, wherein X at position 226 of SEQ ID NO: 93 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP); (w) a β chain comprising the amino acid sequence of SEQ ID NO: 94, wherein X at position 177 of SEQ ID NO: 94 is Ser or Cys (the β chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP). (x) (i) X at position 158 of SEQ ID NO: 116 is Thr or Cys; (ii) X at position 222 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 224 of SEQ ID NO: 116 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) a sequence (y) an α chain comprising the amino acid sequence of SEQ ID NO: 116, in which X at position 225 of SEQ ID NO: 116 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP); (y) a β chain comprising the amino acid sequence of SEQ ID NO: 117, in which X at position 176 of SEQ ID NO: 117 is Ser or Cys (the β chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP). (z) both (v) and (w); (aa) both (x) and (y); (bb) a beta chain comprising the amino acid sequence of SEQ ID NO: 85, wherein X at position 187 of SEQ ID NO: 85 is Ser or Cys (another beta chain of 4360TCR1 with a variant N-terminal signal peptide); (cc) a beta chain comprising the amino acid sequence of SEQ ID NO: 88, wherein X at position 187 of SEQ ID NO: 88 is Ser or Cys (WT (dd) a beta chain comprising the amino acid sequence of SEQ ID NO: 99, wherein X at position 172 of SEQ ID NO: 99 is Ser or Cys (another beta chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP); (ee) both (a) and (bb); (ff) both (b) and (bb); (gg) both (o) and (cc); (hh) both (p) and (cc);(ii) both (v) and (dd); (jj) a β chain comprising the amino acid sequence of SEQ ID NO: 110, wherein X at position 186 of SEQ ID NO: 110 is Ser or Cys (another β chain of 4360TCR5 with a variant N-terminal signal peptide); (kk) a β chain comprising the amino acid sequence of SEQ ID NO: 113, wherein X at position 186 of SEQ ID NO: 113 is Ser or Cys (another β chain of 4360TCR5 with a WT N-terminal signal peptide); (ll) a β chain comprising the amino acid sequence of SEQ ID NO: 122, wherein X at position 171 of SEQ ID NO: 122 is Ser or Cys (another β chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP); (mm) both (d) and (jj); (nn) both (d) and (kk); or (oo) both (x) and (ll). In embodiments of the invention, any one or more of SEQ ID NOs: 21, 22, 41, 42, 55-58, 79, 80, 85, 88, 93, 94, 99, 105, 110, 113, 116, 117, 122, 131, or 134 of the polypeptide are as defined in any one of Tables 2-4;

[0084] Embodiments of the present invention further provide proteins comprising at least one of the polypeptides described herein. By "protein" is meant a molecule comprising one or more polypeptide chains.

[0085] In embodiments, the protein of the present invention may comprise (a) a first polypeptide chain comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 3 and a second polypeptide chain comprising the amino acid sequence of any one of SEQ ID NOs: 4 to 6, or (b) a first polypeptide chain comprising the amino acid sequence of any one of SEQ ID NOs: 31 to 33 and a second polypeptide chain comprising the amino acid sequence of any one of SEQ ID NOs: 34 to 36. The CDR3 of any one or more of SEQ ID NOs: 3, 6, 33, or 36, i.e., the α chain or the β chain, or both, may further comprise a cysteine ​​immediately N-terminal to the first amino acid of the CDR, or a phenylalanine immediately C-terminal to the last amino acid, or both.

[0086] In another embodiment of the invention, the protein comprises (i) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:8; (ii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:129 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:8; (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:63 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:8; (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:130 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:8; (v) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:64; (vi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:129 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:64; (vii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:63 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:64; (viii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:130 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:64. (ix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:65; (x) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:129 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:65; (xi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:63 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:65; (xii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:130 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:65; (xiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66; (xiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:129 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66; (xv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:63 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66; (xvi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:130 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66;(xvii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 38; (xviii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 69; (xix) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 70; (xx) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 71; (xxi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 47 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 48; (xxii) (xxiii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 76; (xxiv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 49 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 50; (xxv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 72 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 73; or (xxvi) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 72 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 102.

[0087] Proteins of the invention may further comprise any of the constant regions described herein with respect to other aspects of the invention. In this regard, in embodiments of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 17, and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 18. In embodiments of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 19, and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 20. In embodiments of the invention, the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 74, and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 75.

[0088] In embodiments of the invention, the protein comprises: (a) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 17, wherein: (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, lie, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is GIy, Ala, Val, Leu, lie, Pro, Phe, Met, or Trp; (b) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) both (a) and (b). In an embodiment of the invention, one or both of the proteins SEQ ID NOs: 17 and 18 are as defined in any one of Tables 2-4.

[0089] Alternatively or additionally, the protein of the present invention may comprise: (a) (i) X at position 179 of SEQ ID NO:21 is Thr or Cys; (ii) X at position 243 of SEQ ID NO:21 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO:21 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO:21 is Gly, Ala, Val, Leu, Ile, Pro, Phe , Met, or Trp; (b) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 21 (the alpha chain of 4360TCR1 with a wild-type N-terminal signal peptide); (i) X at position 180 of SEQ ID NO: 131 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 131 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 131, wherein X at position 247 of SEQ ID NO: 131 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR1 with a wild-type N-terminal signal peptide); (c) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 198 of SEQ ID NO: 22 is Ser or Cys; (ii) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 41 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 41 (the alpha chain of 4360TCR5 with a WT N-terminal signal peptide), wherein: (i) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp;(e) a second polypeptide chain (β chain of 4360TCR5 with a variant N-terminal signal peptide) comprising the amino acid sequence of SEQ ID NO: 42, wherein X at position 197 of SEQ ID NO: 42 is Ser or Cys; (f) both (a) and (c); (g) both (b) and (c); or (h) both (d) and (e); (i) (i) X at position 160 of SEQ ID NO: 55 is Thr or Cys; (ii) X at position 224 of SEQ ID NO: 55 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp. (iii) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 55, wherein X at position 226 of SEQ ID NO: 55 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 55 (the α chain of 4360TCR1 without the N-terminal signal peptide as predicted by IMGT), wherein X at position 227 of SEQ ID NO: 55 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (j) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 56, wherein X at position 173 of SEQ ID NO: 56 is Ser or Cys. a second polypeptide chain comprising the sequence (the β chain of 4360TCR1 without the N-terminal signal peptide as predicted by IMGT); (k) (i) X at position 159 of SEQ ID NO:57 is Thr or Cys; (ii) X at position 223 of SEQ ID NO:57 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO:57 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 226 of SEQ ID NO:57 is (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 57 (the α chain of 4360TCR5 without an N-terminal signal peptide as predicted by IMGT), wherein X at position 172 of SEQ ID NO: 58 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (1) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 58 (the β chain of 4360TCR5 without an N-terminal signal peptide as predicted by IMGT), wherein X at position 172 of SEQ ID NO: 58 is Ser or Cys; (m) both (i) and (j); or (n) both (k) and (1);(o) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 79, wherein (i) X at position 179 of SEQ ID NO: 79 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 79 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 79 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 79 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (p)(i) X at position 180 of SEQ ID NO: 134 is Thr or Cys (ii) X at position 244 of SEQ ID NO: 134 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 134 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 134 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (α chain of 4360TCR1 with a variant N-terminal signal peptide); (q) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 80, wherein X at position 198 of SEQ ID NO: 80 is Ser or Cys (WT (r) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 105, wherein X at position 197 of SEQ ID NO: 105 is Ser or Cys (β chain of 4360TCR5 with a WT N-terminal signal peptide); (s) both (o) and (q); (t) both (p) and (q); (u) both (d) and (r); (v) (i) X at position 159 of SEQ ID NO: 93 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 93 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 93 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp;and (iv) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 93, wherein X at position 226 of SEQ ID NO: 93 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP); (w) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 94, wherein X at position 177 of SEQ ID NO: 94 is Ser or Cys (the α chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP). (i) X at position 158 of SEQ ID NO: 116 is Thr or Cys; (ii) X at position 222 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (iii) X at position 224 of SEQ ID NO: 116 is Met, Ala, Val, Leu, Ile, Pro, Phe, or Trp; and (iv) X at position 224 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, Ile, Pro, Phe, or Trp; (y) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 116, in which X at position 25 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp (the α chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP); (y) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 117, in which X at position 176 of SEQ ID NO: 117 is Ser or Cys (the α chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP). (z) both (v) and (w); (aa) both (x) and (y); (bb) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 85, wherein X at position 187 of SEQ ID NO: 85 is Ser or Cys (another β chain of 4360TCR1 with a variant N-terminal signal peptide); (cc) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 88, wherein X at position 187 of SEQ ID NO: 88 is Ser or Cys (another β chain of 4360TCR1 with a WT N-terminal signal peptide); (dd) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 99, wherein X at position 172 of SEQ ID NO: 99 is Ser or Cys (another β chain of 4360TCR1 without an N-terminal signal peptide as predicted by SignalP);(ee) both (a) and (bb); (ff) both (b) and (bb); (gg) both (o) and (cc); (hh) both (p) and (cc); (ii) both (v) and (dd); (jj) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 110, wherein X at position 186 of SEQ ID NO: 110 is Ser or Cys (another β chain of 4360TCR5 with a variant N-terminal signal peptide); (kk) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 113, wherein X at position 186 of SEQ ID NO: 113 is Ser or Cys (WT (11) an alternative β chain of 4360TCR5 having an N-terminal signal peptide; (11) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 122, in which X at position 171 of SEQ ID NO: 122 is Ser or Cys (an alternative β chain of 4360TCR5 without an N-terminal signal peptide as predicted by SignalP); (mm) both (d) and (jj); (nn) both (d) and (kk); or (oo) both (x) and (11). In embodiments of the invention, any one or more of SEQ ID NOs: 21, 22, 41, 42, 55-58, 79, 80, 85, 88, 93, 94, 99, 105, 110, 113, 116, 117, and 122 are as defined in any one of Tables 2-4.

[0090] The protein of the invention may be a TCR, or, for example, the protein may be a TCR, such as a TCR comprising both SEQ ID NOs: 21 and 22, both SEQ ID NOs: 131 and 22, both SEQ ID NOs: 23 and 24, both SEQ ID NOs: 133 and 24, both SEQ ID NOs: 77 and 78, both SEQ ID NOs: 132 and 78, both SEQ ID NOs: 79 and 80, both SEQ ID NOs: 134 and 80, both SEQ ID NOs: 81 and 82, both SEQ ID NOs: 135 and 82, both SEQ ID NOs: 83 and 84, both SEQ ID NOs: 136 and 84, both SEQ ID NOs: 21 and 85, both SEQ ID NOs: 131 and 85, both SEQ ID NOs: 23 and 87, both SEQ ID NOs: 133 and 87, SEQ ID NOs: A protein of the invention may be a fusion protein if it comprises one polypeptide chain comprising the amino acid sequence of both SEQ ID NOs: 77 and 86, both SEQ ID NOs: 132 and 86, both SEQ ID NOs: 79 and 88, both SEQ ID NOs: 134 and 88, both SEQ ID NOs: 83 and 90, both SEQ ID NOs: 136 and 90, both SEQ ID NOs: 81 and 89, or both SEQ ID NOs: 135 and 89, or if the first and / or second polypeptide chain(s) of the protein further comprises another amino acid sequence, e.g., an amino acid sequence encoding an immunoglobulin or portion thereof. In this regard, embodiments of the invention also provide fusion proteins comprising at least one of the polypeptides of the invention described herein together with at least one other polypeptide. The other polypeptide may be present as a separate protein of the fusion protein, or may be present as a polypeptide expressed in-frame (tandem) with one of the polypeptides of the invention described herein. The other polypeptide may encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to, immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules, e.g., CD1a, CD1b, CD1c, CD1d, etc.

[0091] A fusion protein can include one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein can include 1, 2, 3, 4, 5, or more copies of a polypeptide of the invention and / or another polypeptide. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods.

[0092] In some embodiments of the present invention, the TCRs, polypeptides, and proteins of the present invention may be expressed as a single protein comprising a linker peptide connecting the α chain and the β chain. In this regard, the TCRs, polypeptides, and proteins of the present invention may further comprise a linker peptide. The linker peptide may advantageously facilitate expression of the recombinant TCR, polypeptide, and / or protein in a host cell. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may be a furin-SGSG-P2A linker comprising the amino acid sequence of SEQ ID NO: 25. Once the construct comprising the linker peptide is expressed by a host cell, the linker peptide may be cleaved, resulting in separated α and β chains. In embodiments of the present invention, the TCRs, polypeptides, or proteins may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α chain and the β chain, e.g., α chain-linker-β chain or β chain-linker-α chain.

[0093] In embodiments of the invention, a TCR, polypeptide, or protein may comprise the amino acid sequence set forth in SEQ ID NO: 125, including, from N- to C-terminus, the β chain, the linker (SEQ ID NO: 25), and the α chain. The variant comprises a β chain variable region (with a variant signal peptide) set forth in SEQ ID NO: 8 and a modified β constant domain set forth in SEQ ID NO: 75. The variant full-length β chain is set forth in SEQ ID NO: 78. The variant also comprises an α chain variable region (with a WT signal peptide) set forth in SEQ ID NO: 7 and a modified α constant domain set forth in SEQ ID NO: 74. The variant full-length α chain is set forth in SEQ ID NO: 77.

[0094] In another embodiment of the invention, the TCR, polypeptide, or protein may comprise the amino acid sequence set forth in SEQ ID NO: 126, including, from N- to C-terminus, the alpha chain, the linker (SEQ ID NO: 25), and the beta chain. The variant comprises an alpha chain variable region (with a variant signal peptide) set forth in SEQ ID NO: 63 and a modified alpha constant domain set forth in SEQ ID NO: 74. The variant full-length alpha chain is set forth in SEQ ID NO: 81. The variant also comprises a beta chain variable region (with a WT signal peptide) set forth in SEQ ID NO: 64 and a modified beta constant domain set forth in SEQ ID NO: 75. The variant full-length beta chain is set forth in SEQ ID NO: 82.

[0095] In embodiments of the invention, a TCR, polypeptide, or protein may comprise the amino acid sequence set forth in SEQ ID NO: 127, including, from N- to C-terminus, the beta chain, the linker (SEQ ID NO: 25), and the alpha chain. A variant comprises a beta chain variable region (with a variant signal peptide) set forth in SEQ ID NO: 38 and a modified beta constant domain set forth in SEQ ID NO: 75. A variant full-length beta chain is set forth in SEQ ID NO: 104. A variant also comprises an alpha chain variable region set forth in SEQ ID NO: 37 and a modified alpha constant domain set forth in SEQ ID NO: 74. A variant full-length alpha chain is set forth in SEQ ID NO: 103.

[0096] In another embodiment of the invention, the TCR, polypeptide, or protein may comprise the amino acid sequence set forth in SEQ ID NO: 128, including, from N- to C-terminus, the alpha chain, the linker (SEQ ID NO: 25), and the beta chain. The variant comprises an alpha chain variable region set forth in SEQ ID NO: 37 and a modified alpha constant domain set forth in SEQ ID NO: 74. The full-length alpha chain of the variant is set forth in SEQ ID NO: 103. The variant also comprises a beta chain variable region (with WT signal peptide) set forth in SEQ ID NO: 70 and a modified beta constant domain set forth in SEQ ID NO: 75. The full-length beta chain of the variant is set forth in SEQ ID NO: 106.

[0097] In embodiments of the invention, the TCR, polypeptide, or protein may comprise an alternative amino acid sequence set forth in SEQ ID NO: 137, including, from N- to C-terminus, a beta chain, a linker (SEQ ID NO: 25), and an alpha chain. The variant comprises a beta chain variable region (with a variant signal peptide) set forth in SEQ ID NO: 8 and a modified beta constant domain set forth in SEQ ID NO: 75. The variant full-length beta chain is set forth in SEQ ID NO: 78. The variant also comprises an alpha chain variable region set forth in SEQ ID NO: 129 and a modified alpha constant domain set forth in SEQ ID NO: 74. The variant full-length alpha chain is set forth in SEQ ID NO: 132.

[0098] In another embodiment of the invention, the TCR, polypeptide, or protein may comprise an alternative amino acid sequence set forth in SEQ ID NO: 138, including, from N- to C-terminus, an alpha chain, a linker (SEQ ID NO: 25), and a beta chain. The variant comprises an alternative alpha chain variable region set forth in SEQ ID NO: 130 and a modified alpha constant domain set forth in SEQ ID NO: 74. The full-length alpha chain of the variant is set forth in SEQ ID NO: 135. The variant also comprises a beta chain variable region (with WT signal peptide) set forth in SEQ ID NO: 64 and a modified beta constant domain set forth in SEQ ID NO: 75. The full-length beta chain of the variant is set forth in SEQ ID NO: 82.

[0099] In some embodiments, a TCR, polypeptide, or protein disclosed herein comprises an α chain and / or a β chain disclosed herein, including a signal peptide. In some embodiments, the sequence of the signal peptide of any of the α chain and / or β chain disclosed herein comprises an alanine or histidine residue substituted for the wild-type residue at position 2.

[0100] In some embodiments, the TCRs, polypeptides, or proteins disclosed herein comprise mature versions of the alpha and / or beta chains disclosed herein that lack the signal peptide. The sequence of the signal peptide or the mature forms of the alpha and / or beta chains can be performed according to any method known in the art, including IMGT and SignalP.

[0101] The protein of the present invention may be a recombinant antibody or antigen-binding portion thereof comprising at least one of the polypeptides of the present invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the present invention and an antibody polypeptide chain or antigen-binding portion thereof. The antibody polypeptide or antigen-binding portion thereof may be an antibody heavy chain, light chain, heavy or light chain variable or constant region, single-chain variable fragment (scFv), or Fc, Fab, or F(ab)2' fragment, etc. The antibody polypeptide chain or antigen-binding portion thereof may exist as separate polypeptides in the recombinant antibody. Alternatively, the antibody polypeptide chain or antigen-binding portion thereof may exist as a polypeptide expressed in frame (tandem) with a polypeptide of the present invention. The antibody polypeptide or antigen-binding portion thereof may be any antibody polypeptide or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0102] Functional variants of the TCRs, polypeptides, or proteins of the present invention described herein are included within the scope of the present invention. The term "functional variant," as used herein, refers to a TCR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, and the functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants include, for example, variants of the TCRs, polypeptides, or proteins described herein (parent TCRs, polypeptides, or proteins) that retain, to a similar, identical, or greater extent than the parent TCR, polypeptide, or protein, the antigen specificity of the parent TCR, or the ability to specifically bind to a mutant RAS to which the parent polypeptide or protein specifically binds. In relation to a parent TCR, polypeptide, or protein, a functional variant may, for example, be at least about 30%, about 50%, about 75%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent TCR, polypeptide, or protein, respectively.

[0103] A functional variant may, for example, comprise the amino acid sequence of a parent TCR, polypeptide, or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, conservative amino acid substitutions may be substitutions of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), an amino acid having a nonpolar side chain with an amino acid having a different nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid with another basic amino acid (e.g., Lys, Arg), an amino acid having a polar side chain with an amino acid having a different polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0104] Alternatively, or in addition, a functional variant may comprise the amino acid sequence of a parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide, or protein.

[0105] The signal peptide of each of the TCRs, polypeptides, proteins, functional variants, and functional portions described herein, when present, may be any suitable TCR signal peptide, so long as the TCR, polypeptide, protein, or functional variant is expressed and has antigen specificity for a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with valine as presented by HLA class II molecules.

[0106] A TCR, polypeptide, or protein may consist essentially of a specified amino acid sequence(s) described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not substantially alter the biological activity of the TCR, polypeptide, or protein. In this regard, a TCR, polypeptide, or protein of the present invention may consist essentially of the amino acid sequence of, for example, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, both SEQ ID NOs:21-22, or both SEQ ID NOs:23-24, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:39, SEQ ID NO:40, both SEQ ID NOs:41-42, or both SEQ ID NOs:39-40. Also, for example, a TCR, polypeptide, or protein of the present invention may consist essentially of the amino acid sequence(s) of (i) SEQ ID NO:7, (ii) SEQ ID NO:8, (iii) SEQ ID NO:37, (iv) SEQ ID NO:38, (v) both SEQ ID NOs:7 and 8, or (vi) both SEQ ID NOs:37 and 38. Furthermore, the TCRs, polypeptides, or proteins of the invention may consist essentially of the amino acid sequences of (a) any one or more of SEQ ID NOs: 1-6 and 31-36; (b) all of SEQ ID NOs: 1-3; (c) all of SEQ ID NOs: 4-6; (d) all of SEQ ID NOs: 31-33; (e) all of SEQ ID NOs: 34-36; (f) all of SEQ ID NOs: 1-6; or (g) all of SEQ ID NOs: 31-36.

[0107] The TCRs, polypeptides, and proteins of the present invention may be of any length, i.e., contain any number of amino acids, so long as the TCR, polypeptide, or protein retains its biological activity, such as the ability to specifically bind to mutant RAS, detect cancer in a mammal, or treat or prevent cancer in a mammal. For example, polypeptides may range from about 50 to about 5,000 amino acids in length, e.g., about 50, about 70, about 75, about 100, about 125, about 150, about 175, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1,000, or more amino acids in length. In this regard, polypeptides of the present invention also include oligopeptides.

[0108] The TCRs, polypeptides, and proteins of the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline, 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0109] The TCRs, polypeptides or proteins of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or multimerized or conjugated.

[0110] The TCRs, polypeptides, and / or proteins of the invention can be obtained by methods known in the art, for example, de novo synthesis. Polypeptides and proteins can also be produced recombinantly using standard recombinant methods and the nucleic acids described herein. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein may be commercially synthesized by a commercial entity. In this regard, the TCRs, polypeptides, and proteins of the invention may be synthetic, recombinant, isolated, and / or purified. An embodiment of the invention provides an isolated or purified TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein with respect to other aspects of the invention. Another embodiment of the invention provides an isolated or purified TCR, polypeptide, or protein resulting from the intracellular expression of any of the nucleic acids or vectors described herein with respect to other aspects of the invention. Yet another embodiment of the invention provides a method of producing any of the TCRs, polypeptides, or proteins described herein, comprising culturing any of the host cells or populations of host cells described herein, such that the TCR, polypeptide, or protein is produced.

[0111] Also included within the scope of the present invention are conjugates, e.g., bioconjugates, comprising any of the TCRs, polypeptides, or proteins (including any functional portion or variant thereof), nucleic acids, recombinant expression vectors, host cells, populations of host cells, or antibodies or antigen-binding portions thereof of the present invention. Conjugates, and generally methods for synthesizing conjugates, are known in the art.

[0112] Embodiments of the present invention provide nucleic acids comprising nucleotide sequences encoding any of the TCRs, polypeptides, or proteins described herein. As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule" and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, e.g., phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In embodiments, nucleic acids include complementary DNA (cDNA). It is generally preferred that nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, it may be preferable for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions, as discussed herein.

[0113] Preferably, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments into a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule obtained by replication of the above (i). For purposes herein, replication may be in vitro or in vivo.

[0114] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6 Examples of nucleic acids include, but are not limited to, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queusine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from a commercial entity.

[0115] The nucleic acid may comprise any nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. In embodiments of the invention, the nucleic acid may comprise the nucleotide sequence of any one of SEQ ID NOs: 43-46 (Table 5). In embodiments of the invention, the nucleic acid comprises the nucleotide sequence of both SEQ ID NOs: 43-44 or both amino acid sequences of SEQ ID NOs: 45-46.

[0116] [Table 5]

[0117] In embodiments of the present invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound by any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence increases the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence can involve replacing native codons with alternative codons that encode the same amino acid but that can be translated by more readily available tRNAs in the cell, thereby increasing translation efficiency. Optimizing a nucleotide sequence can also reduce secondary structures in the mRNA that interfere with translation, thereby increasing translation efficiency.

[0118] The present invention also provides nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of any of the nucleic acids described herein, or a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0119] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably greater than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or those containing only a few scattered mismatches, from random sequences that happen to have a few small regions (e.g., 3-10 bases) of nucleotide sequence match. Such small regions of complementarity melt more easily than full-length complements of 14-17 bases or more, making them readily distinguishable by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent and temperatures of about 50-70°C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the TCRs of the present invention. It will generally be appreciated that conditions can be made more stringent by adding increasing amounts of formamide.

[0120] The invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid can consist essentially of any of the nucleotide sequences described herein.

[0121] An embodiment of the present invention is an isolated or purified nucleic acid comprising, in 5' to 3' direction, a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences are, respectively, 7 and 8; 7 and 64; 63 and 8; 63 and 64; 7 and 65; 63 and 65; 7 and 66; 63 and 66; 8 and 7; 64 and 7; 8 and 63; 64 and 63; 65 and 7; 65 and 63; 66 and 7; 66 and 63; 129 and 8; 129 and 64; 129 and 65; 129 and 66; 8 and 129; 64 and 129; 65 and 129; 66 and 129; 130 and 8;130 and 64;130 and 65;130 and 66;8 and 130;64 and 130;65 and 130;66 and 130;37 and 38;37 and 69;37 and 70;37 and 71;38 and 37;69 and 37;70 and 37;71 and 37;23 and 24;23 and 84;83 and 24;83 and 84;23 and 87;83 and 87;23 and 90;83 and 90;24 and 23;84 and 23;24 and 83;84 and 83;87 and 23;87 and 83;90 and 23;90 and 83;133 and 24;133 and 84;133 and 87 ;133 and 90;24 and 133;84 and 133;87 and 133;90 and 133;39 and 40;39 and 107;39 and 112;39 and 115;40 and 39;107 and 39;112 and 39;115 and 39;136 and 24;136 and 84;136 and 87;136 and 90;24 and 136;84 and 136;87 and 136;90 and 136;21 and 22;21 and 80;79 and 22;79 and 80;21 and 85;21 and 88;79 and 85;79 and 88;22 and 21;80 and 21;22 and 79;80 and 7 9;85 and 21;88 and 21;85 and 79;88 and 79;131 and 22;131 and 80;131 and 85;131 and 88;22 and 131;80 and 131;85 and 131;88 and 131;134 and 22;134 and 80;134 and 85;134 and 88;22 and 134;80 and 134;85 and 134;88 and 134;77 and 78;77 and 82;81 and 78;81 and 82;77 and 86;81 and 86;78 and 77;82 and 77;78 and 81;82 and 81;86 and 77;86 and 81;132 and 78;132 and 82; 132 and 86; 78 and 132; 82 and 132; 86 and 132; 135 and 78; 135 and 82; 135 and 86; 78 and 135; 82 and 135; 86 and 135; 77 and 89; 81 and 89; 89 and 77; 89 and 81; 132 and 89; 89 and 132; 135 and 89; 89 and 135; 41 and 42; 41 and 105; 41 and 11 0;41 and 113;42 and 41;105 and 41;110 and 41;113 and 41;103 and 104;103 and 111;103 and 114;104 and 103;111 and 103;114 and 103;103 and 106;106 and 103;47 and 48;48 and 47;67 and 68;67 and 76;68 and 67;76 and 67;49 and 50;50 and 49; 72 and 73;72 and 102;73 and 72;102 and 72;51 and 52;52 and 51;53 and 54;54 and 53;55 and 56;56 and 55;57 and 58;58 and 57;91 and 92;92 and 91;108 and 109;109 and 108;93 and 94;93 and 99;94 and 93;99 and 93;97 and 98;97 and 101;98 and 97;1 Nucleic acids encoding the amino acid sequences of 01 and 97; 95 and 96; 95 and 100; 96 and 95; 100 and 95; 116 and 117; 116 and 122; 117 and 116; 122 and 116; 120 and 121; 120 and 124; 121 and 120; 124 and 120; 118 and 119; 118 and 123; 119 and 118, or 123 and 118 are provided.

[0122] In an embodiment of the invention, the isolated or purified nucleic acid further comprises a third nucleotide sequence interposed between the first and second nucleotide sequences, wherein the third nucleotide sequence encodes a cleavable linker peptide. In an embodiment of the invention, the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO:25.

[0123] The nucleic acids of the present invention can be incorporated into recombinant expression vectors. In this regard, the present invention provides recombinant expression vectors comprising any of the nucleic acids of the present invention. In embodiments of the present invention, the recombinant expression vector comprises nucleotide sequences encoding the α chain, the β chain, and the linker peptide.

[0124] For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and enables a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with the cell under conditions sufficient to express the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present invention are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring internucleotide linkages, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

[0125] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation, expression, or both, such as plasmids and viruses. Vectors may be selected from the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a viral vector, such as a retroviral vector. In a particularly preferred embodiment, the recombinant expression vector is an MSGV1 vector. In an embodiment of the invention, the recombinant expression vector is a transposon or lentiviral vector.

[0126] The recombinant expression vectors of the present invention can be prepared using standard recombinant DNA techniques, for example, as described in Green and Sambrook et al., supra. Expression vector constructs, either circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0127] Desirably, the recombinant expression vector includes regulatory sequences, e.g., transcription and translation initiation and termination codons, as appropriate, and considering whether the vector is DNA- or RNA-based, specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced.

[0128] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to, e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to confer prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0129] The recombinant expression vector may comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or to a nucleotide sequence complementary to or hybridizing to a nucleotide sequence encoding a TCR, polypeptide, or protein. For example, selection of strong, weak, inducible, tissue-specific, and developmental stage-specific promoters is within the skill of one of ordinary skill in the art. Similarly, combinations of nucleotide sequences and promoters are also within the skill of one of ordinary skill in the art. The promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter, and promoters found in the long terminal repeat of murine stem cell virus.

[0130] The recombinant expression vectors of the invention can be designed for transient expression, stable expression, or both. The recombinant expression vectors can also be made for constitutive or inducible expression.

[0131] Additionally, the recombinant expression vector may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of cells expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, on the cell in which the gene is expressed, causing the cell to die when contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0132] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell capable of containing a recombinant expression vector of the present invention. Host cells may be eukaryotic cells, such as plants, animals, fungi, or algae, or prokaryotic cells, such as bacteria or protozoa. Host cells may be cultured cells or primary cells, i.e., cells isolated directly from an organism, such as a human or mouse. Host cells may be adherent cells or suspension cells, i.e., cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be of any cell type, may be derived from any type of tissue, and may be at any stage of development, but the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. In an embodiment of the invention, the host cell is a human lymphocyte. In another embodiment of the invention, the host cell is selected from a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, and a natural killer (NK) cell. Yet another embodiment of the invention provides a method of generating a host cell expressing a TCR having antigen-specificity for the peptide of SEQ ID NO: 30, comprising contacting the cell with any of the vectors described herein under conditions that allow the vector to be introduced into the cell.

[0133] For purposes herein, a T cell may be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell may also be enriched or purified. Preferably, the T cell is a human T cell. The T cell may be any type of T cell, may be at any stage of development, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0134] Also provided by the present invention are populations of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any of the recombinant expression vectors, or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogeneous population comprising primarily host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population may also be a clonal population of cells, in which all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vectors described herein.

[0135] In embodiments of the present invention, the number of cells within a population can be rapidly expanded. Expansion of T cells can be achieved by any of a number of methods known in the art, for example, as described in U.S. Patent Nos. 8,034,334, 8,383,099, U.S. Patent Application Publication No. 2012 / 0244133, Dudley et al., J. Immunother., 26:332-42 (2003), and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). In embodiments, expansion of T cells is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0136] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term "isolated," as used herein, means removed from its natural environment. The term "purified," as used herein, means increased purity, although "purity" is a relative term and is not necessarily to be construed as absolute purity. For example, purity may be at least about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or even about 100%.

[0137] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the invention (collectively hereinafter referred to as the "TCR materials of the invention") may be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention may include more than one TCR material of the invention, e.g., a polypeptide and nucleic acid, or two or more different TCRs. Alternatively, the pharmaceutical composition may include the TCR material of the invention in combination with another pharmaceutically active agent(s) or drug(s), e.g., a chemotherapeutic agent, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0138] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the invention under consideration. Methods of preparing administrable compositions will be known or apparent to those skilled in the art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 22 nd Ed., Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0139] The choice of carrier will be determined in part by the specific TCR material of the invention, as well as by the specific method used to administer the TCR material of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the invention. Suitable formulations may include those administered parenterally, subcutaneously, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, or intraperitoneally. More than one route may be used to administer the TCR material of the invention, and in certain instances, a particular route may provide a more immediate and effective response than another route.

[0140] Preferably, the TCR material of the present invention is administered, for example, by intravenous injection. When the TCR material of the present invention is a host cell (or population thereof) expressing a TCR of the present invention, a pharmaceutically acceptable carrier for the cells for injection can include any isotonic carrier, such as normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0141] For purposes of the present invention, the amount or dose (e.g., number of cells if the TCR material of the present invention is one or more cells) of the TCR material of the present invention administered should be sufficient to produce, e.g., a therapeutic or prophylactic response in the subject or animal over an appropriate time frame. For example, a dose of the TCR material of the present invention should be sufficient to bind to a cancer antigen (e.g., mutant RAS) or detect, treat, or prevent cancer for a period of about 2 hours or more from the time of administration, e.g., 12-24 hours or more. In certain embodiments, the period may be even longer. The dose will be determined by the efficacy of the particular TCR material of the present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) being treated.

[0142] Many assays for determining the dose to be administered are known in the art. For purposes of the present invention, a starting dose to be administered to a mammal can be determined using, for example, an assay that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a given dose of the TCR, polypeptide, or protein of the present invention when administered to a mammal, among a set of mammals that have each received different doses of T cells. The extent to which target cells are lysed or IFN-γ is secreted when a particular dose is administered can be assayed by methods known in the art.

[0143] The dose of the TCR material of the present invention will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR material of the present invention. Typically, the attending physician will determine the dose of the TCR material of the present invention for treating each individual patient, taking into account various factors, e.g., age, weight, general health, diet, sex, the TCR material of the present invention to be administered, the route of administration, and the severity of the cancer being treated. In embodiments where the TCR material of the present invention is a population of cells, the number of cells administered per infusion may be, for example, about 1 x 10 6 ~Approx. 1×10 12 In certain embodiments, the number of cells may vary from 1 x 10 to 1 x 10. 6 Subcellular amounts may also be administered.

[0144] Those skilled in the art will readily appreciate that the TCR materials of the present invention may be modified in any number of ways, thereby increasing the therapeutic or prophylactic efficacy of the TCR materials of the present invention through such modifications. For example, the TCR materials of the present invention may be conjugated to a chemotherapeutic agent, either directly or indirectly via cross-linking. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art will recognize that sites on the TCR materials of the present invention that are not essential to the function of the TCR materials of the present invention are suitable sites for conjugating cross-linking and / or chemotherapeutic agents, so long as the cross-linking and / or chemotherapeutic agent does not interfere with the function of the TCR materials of the present invention, i.e., their ability to bind mutant RAS or detect, treat, or prevent cancer, once bound to the TCR materials of the present invention.

[0145] It is contemplated that the pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and populations of cells of the invention can be used in methods of treating or preventing cancer. Without being bound by theory, it is believed that the TCRs of the invention specifically bind to mutant RAS, such that, when expressed by a cell, the TCR (or related polypeptides or proteins of the invention) can mediate an immune response against target cells expressing mutant RAS. In this regard, the invention provides a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0146] Embodiments of the present invention provide a method of inducing an immune response against cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to induce an immune response against cancer in the mammal.

[0147] Embodiments of the present invention provide any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0148] Embodiments of the present invention provide any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, for use in inducing an immune response against cancer in a mammal.

[0149] The terms "treat" and "prevent," as well as derivatives thereof, as used herein, do not necessarily mean 100%, i.e., complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any amount or level of cancer treatment or prevention in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting tumor regression. Also, for purposes herein, "prevention" can include delaying the onset of cancer or a symptom or condition thereof. Alternatively, or in addition, "prevention" can include preventing or delaying the recurrence of cancer or a symptom or condition thereof.

[0150] Also provided is a method for detecting the presence of cancer in a mammal, the method comprising (i) contacting any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions of the invention described herein with a sample comprising one or more cells from a mammal, thereby forming a complex, and (ii) detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0151] For the methods of the present invention for detecting cancer in a mammal, the cellular sample may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, such as a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0152] For purposes of the present method of detecting cancer, contacting may be performed in vitro or in vivo in a mammal, preferably in vitro.

[0153] Detection of the complex can also be achieved through any number of methods known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells of the invention described herein may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle).

[0154] For purposes of the methods of the present invention in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0155] In the context of the methods of the present invention, cancer may be, for example, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, or gastrointestinal carcinoid tumor. The cancer may be any cancer, including glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharynx cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. Preferred cancers are pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, and the pancreatic cancer is pancreatic adenocarcinoma. In embodiments of the invention, the cancer expresses a mutant human RAS amino acid sequence, which is a mutant human KRAS, a mutant human HRAS, or a mutant human NRAS amino acid sequence. The mutant human KRAS, HRAS, and NRAS expressed by the cancer may be as described herein with respect to other aspects of the invention.

[0156] The mammal referred to in the methods of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, such as mice and hamsters, and lagomorph mammals, such as rabbits. Preferably, the mammal is a mammal of the order Carnivora, which includes cats and dogs. More preferably, the mammal is a mammal of the order Artiodactyla, which includes cattle and pigs, or Perissodactyla, which includes horses. Most preferably, the mammal is a mammal of the order Primates, Ceboids or Simoids (monkeys), or Anthropoids (humans and apes). A particularly preferred mammal is a human.

[0157] It should be noted that the above are merely exemplary embodiments. Other exemplary embodiments will become apparent from the entire description herein. Those skilled in the art will also understand that each of these embodiments may be used in various combinations with the other embodiments provided herein.

[0158] The following examples further illustrate the present invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0159] Example 1 This example is RAS G12V 1 shows the identification of peripheral blood lymphocytes (PBLs) reactive to

[0160] PBLs were sorted into CD4 or CD8 memory and effector T cells.

[0161] In vitro stimulation (IVS) was performed separately on PBLs from patient 4360 enriched for CD4 or CD8 cells. 10 μg / mL RAS G12V PBLs were stimulated with DCs loaded with long peptide (LP) (MTEYKLVVVGAVGVGKSALTIQLI, SEQ ID NO: 30). After 2 weeks of stimulation, T cells were restimulated and FACS sorted. Figure 1A shows that CD4 T lymphocytes were stimulated by RAS G12V Dot plots of flow cytometry assays showing the gating strategy are shown. DCs were sorted for high expression of OX40 and 41BB surface markers after LP IVS (DMSO-treated DCs were used as a negative control). Sorted cells were then sorted and expanded using rapid expansion (REP) for 14 days, as shown in Figure 1A.

[0162] The expanded cells were then treated with 10 μg / mL RAS G12VDCs were stimulated with LP-loaded or DMSO-treated DCs, and CD4 T lymphocytes highly expressing OX40 and 41BB surface markers were then sorted into 96-well plates for single-cell sequencing, as shown in Figure 1B.

[0163] After the IVS, the cells were transfected with either WT / mutant tandem minigene (TMG) or 1 μg / mL of RAS. G12V LP (SEQ ID NO: 30) or RAS WT Responsiveness to RAS was tested by co-culturing DCs loaded with LP (MTEYKLVVVGAGGVGKSALTIQLI, SEQ ID NO: 27) or treated with an equal volume of DMSO. Cells stimulated and then expanded with REP but not sorted as described above and as in Figure 1A ("unsorted" cells) or cells grown with IL2 alone but not stimulated with RAS antigen-bearing DCs ("no IVS" cells) served as controls. T cells with and without anti-CD28 / CD3 beads also served as negative and positive controls, respectively. After overnight co-culture, cells were analyzed by IFN-γ ELISpot (Figure 1C) to determine the live / CD3 activity. + / CD4 + were analyzed by flow cytometry for upregulation of the 41BB / OX40 surface marker in the gated population (Figure 1D).

[0164] Example 2 This example illustrates the characterization of the PBLs of Example 1.

[0165] As shown in 84.2% of the cells in Figure 1B, RAS G12V After LP IVS (including one REP after sorting), the test was performed on the CD4 PBL cells of Example 1. The cells were treated with various concentrations of RAS G12V LP or RAS WT After overnight co-culture, the cells were analyzed by IFN-γ ELISpot (Figure 2A) and flow cytometry (Figure 2B, Live / CD3 + / CD4 + RAS was analyzed using the 41BB / OX40 surface marker upregulation in the gated population. G12V Strong avidity for LP was observed (down to approximately 10 pg / mL LP).

[0166] Using IFN-γ ELISpot (Figure 3, left axis and bars) and 41BB / OX40 flow cytometry assays (Figure 3, right axis and circles), we investigated the RAS G12V We identified the MHC-II restriction elements recognized by CD4 PBLs undergoing LP IVS. We transfected them with DNA plasmids containing various combinations of patient MHC-II α and β chains and expressed RAS. G12V The cells were co-cultured with COS7 cells loaded with LP. RAS was restricted by DPB1*03:01. G12V Cellular reactivity was observed against

[0167] Example 3 This example illustrates the identification of TCRs of PBLs of Example 2, according to an embodiment of the present invention.

[0168] Two TCRs (TCR1, TCR2) identified by single-cell sequencing were sequenced from CD4 PBLs (within 84.2% of cells, as shown in Figure 1B and described in Example 1) of patient 4360 after LP IVS.

[0169] Table 6 shows the sequence of 4360TCR1, with the CDR sequences underlined.

[0170] [Table 6-1]

[0171] [Table 6-2]

[0172] [Table 6-3]

[0173] TCR2 was found to have a CDR3β sequence of ASSSGTGVAEAF (SEQ ID NO: 26), which also had a cysteine ​​N-terminal to the first amino acid (alanine) and a phenylalanine C-terminal to the last amino acid (phenylalanine).

[0174] Deep sequencing (Adaptive Biotechnologies, Seattle, WA, USA) of DNA extracts from four tumor fragments revealed that TCR1 was present in one of these fragments (5.7 repeats per 100,000 cells), but TCR2 was absent from any of the fragments.

[0175] Example 4 This example demonstrates the construction of retroviral vectors encoding TCR1 and TCR2 according to an embodiment of the present invention.

[0176] An MSGV1-based retroviral vector was constructed encoding the variable regions of the TCR alpha and beta chains of TCR1, except that the amino acid residue at position 2 of the N-terminal signal peptide of the beta chain was changed to alanine to facilitate cloning into the vector. Additional modifications were made to the wild-type TCR, as described in more detail below.

[0177] A CD22-specific TCR was constructed as previously described (Jin et al., JCI Insight, 3(8):e99488 (2018), incorporated herein by reference in its entirety). Briefly, the TCRβ VDJ region was fused to a murine TCRβ constant chain, and the TCRα VJ region was fused to a murine TCRα constant chain. Without being bound by any particular theory or mechanism, it is believed that the use of murine constant regions improves TCR expression and functionality (Cohen et al., Cancer Res., 66(17):8878-8886 (2006)).

[0178] Furthermore, we modified the mouse TCRα and TCRβ constant chains with cysteine ​​residues and introduced transmembrane hydrophobic mutations into the mouse TCRα constant chain. Without being bound by any particular theory or mechanism, we believe that these modifications preferentially pair the introduced TCR chains, enhancing TCR surface expression and functionality (Cohen et al., Cancer Res., 67(8):3898-903 (2007); Haga-Friedman et al., J. Immun., 188:5538-5546 (2012)).

[0179] To ensure equal expression efficiency of the two chains, the TCR β chain and the TCR α chain were separated by a furin SGSG P2A linker (RAKRSGSGATNFSLKQAGDVEENPGP) (SEQ ID NO: 25) (Szymczak et al., Nat. Biotechnol., 22(5):589-94 (2004)).

[0180] The second amino acid of the TCR Vβ chain (the second amino acid in the N-terminal signal peptide) was changed to alanine (A) to allow cloning of the TCR expression cassette into the 5' NcoI site of the MSGV1 vector. The expression cassette had the following configuration: 5' NcoI-VDJβ-mCβ-furin / SGSG / P2A-VJα-mCα-SalI3'. The TCR nucleotide sequence was codon-optimized for expression in human T cells using the Genscript codon optimization tool. This example describes the synthesis of a bicistronic vector with a 5' TCRβ→TCRα3' orientation, although the TCRβ→TCRα order can also be reversed. The vector insert sequence was codon-optimized for expression in human tissues.

[0181] Example 5 This example illustrates the characterization of the TCR of PBLs of Example 2 identified in Example 3 using the retroviral vector of Example 4, according to an embodiment of the present invention.

[0182] TCR1 and TCR2 were each virally transduced into a Jurkat-CD4-NFAT-luciferase cell line, followed by transfection with 1 μg / mL of RAS. G12V LP, RAS WT DCs were co-cultured with LP-loaded DCs or DCs treated with an equal volume of DMSO, and luciferase activity was measured (Fig. 4A).

[0183] PBLs from patient 4360 were transduced with TCR1 or TCR2, or, as negative controls, transduced with WT GFP, empty plasmid (mock), or left untransduced. PBLs after LP IVS (Example 1, Figures 1 and 2) were used as positive controls. Cells were then transduced with RAS G12V LP or RAS WT Co-cultured with autologous DCs loaded with LP or RAS G12V Full length (FL) (SEQ ID NO: 14) or RAS WT The PBLs were co-cultured with autologous DC mRNA transfected with FL (SEQ ID NO: 10). T cells cultured alone and PBLs cultured with DMSO were used as negative controls. PBLs activated with Dynabeads conjugated to anti-CD3 / anti-CD28 antibodies were used as positive controls. PBLs after LP IVS were also used as positive controls. CD3 + / CD8 + Gated cells (Figure 4B) or CD3 + / CD4 + TCR reactivity was assessed by flow cytometry assay for 4-1BB and OX40 (% 4-1BB+ / OX40+) expression in gated cells (Figure 4C).

[0184] Example 6 In this example, TILs after IVS were analyzed using RAS G12V These results indicate that the antibody is reactive to TCR1 and recognizes the same MHC-II restriction as TCR1.

[0185] TIL fragments were used as a cell source and stimulated with IVS. TILs were then transfected with RAS G12VCo-cultured with autologous DCs pulsed with LP peptide or RAS G12V The cells were co-cultured with RNA-transfected autologous DCs in FL. If there were not enough cells present at any stage of IVS, some fragments were pooled with other fragments. T cells (TILs) co-cultured alone and TILs co-cultured with DMSO-loaded DCs were used as negative controls. TILs cultured with Dynabeads conjugated to anti-CD3 / anti-CD28 antibodies served as positive controls. The TILs were tested for reactivity after IVS by measuring the expression of 41BB and OX40 by flow cytometry (Figure 5A) and by IFN-γ ELISPOT assay for IFN-γ secretion (Table 7).

[0186] [Table 7]

[0187] The MHC-II restriction elements recognized by 4360 CD4 TILs after IVS were determined using IFN-γ ELISpot. Patients were transfected with DNA plasmids containing various combinations of MHC-II α and β chains, and RAS G12V The cells were co-cultured with COS7 cells loaded with LP. PBLs virally transduced with TCR1 were used as a positive control. The results are shown in Figure 5B. After IVS, TILs were found to recognize the same MHC-II restriction as TCR1.

[0188] Example 7 This example demonstrates that the TCR found in TILs persists in tumor fragments.

[0189] Using single-cell sequencing, six additional TCR sequences were found in TILs after IVS as described in Example 6 (TCR5-10).

[0190] Table 8 shows the sequence of 4360TCR5, with the CDR sequences underlined.

[0191] [Table 8-1]

[0192] [Table 8-2]

[0193] TCR6 to 10 were found to have the following CDR3β sequences: TCR6: ASTLQGRAGANVLT (SEQ ID NO: 29); TCR7: ASSQPGLAGGGDTQY (SEQ ID NO: 59); TCR8: ASSQSTSGSGSSIQY (SEQ ID NO: 60); TCR9: ATSRDVGSVEQY (SEQ ID NO: 61); TCR10: ASSPNAGNTEAF (SEQ ID NO: 62). Furthermore, TCR5 to 10 were found to have a cysteine ​​on the N-terminal side of the first amino acid (serine / alanine), and TCR5 to 9 were found to have a phenylalanine on the C-terminal side of the last amino acid (tyrosine / phenylalanine).

[0194] Deep sequencing results of four different tumor fragments (FrTu) from patient 4360 show that TCR5 is present in all fragments tested, TCR6, 8, and 9 are present in one fragment, TCR7 is present in two fragments, and TCR10 is absent from all fragments.

[0195] TCR virally transduced PBLs were then transduced into RAS G12V or RAS WT Reactivity was tested by co-culturing with autologous DCs loaded with LP. DMSO-loaded DCs served as a negative control. PBLs cultured with Dynabeads conjugated to anti-CD3 / anti-CD28 antibodies served as a positive control. IFN-γ ELISPOT assays were performed (Figure 6). No reactivity was detected with TCR6, 7, 9, or 10. TCR8 showed reactivity in all experiments and was not pursued further.

[0196] Example 8 This example demonstrates the construction of a retroviral vector encoding TCR5 according to an embodiment of the present invention.

[0197] An MSGV1-based retroviral vector was constructed encoding the variable regions of the TCR alpha and beta chains of TCR1, except that the amino acid residue at position 2 of the N-terminal signal peptide of the beta chain was changed to alanine to facilitate cloning into the vector. Additional modifications were made to the wild-type TCR, as described in more detail below.

[0198] A CD22-specific TCR was constructed as previously described (Jin et al., JCI Insight, 3(8):e99488 (2018), incorporated herein by reference in its entirety). Briefly, the TCRβ VDJ region was fused to a murine TCRβ constant chain, and the TCRα VJ region was fused to a murine TCRα constant chain. Without being bound by any particular theory or mechanism, it is believed that the use of murine constant regions improves TCR expression and functionality (Cohen et al., Cancer Res., 66(17):8878-8886 (2006)).

[0199] Furthermore, we modified the constant chains of mouse TCRα and TCRβ with cysteines and introduced transmembrane hydrophobic mutations into the mouse TCRα constant chain. Without being bound by any particular theory or mechanism, we believe that these modifications preferentially pair the introduced TCR chains, enhancing TCR surface expression and functionality (Cohen et al., Cancer Res., 67(8):3898-903 (2007); Haga-Friedman et al., J. Immu., 188:5538-5546 (2012)).

[0200] To ensure equal expression efficiency of the two chains, the TCR β chain and the TCR α chain were separated by a furin SGSG P2A linker (RAKRSGSGATNFSLKQAGDVEENPGP) (SEQ ID NO: 25) (Szymczak et al., Nat. Biotechnol., 22(5):589-94 (2004)).

[0201] The second amino acid of the TCR Vβ chain (the second amino acid in the N-terminal signal peptide) was changed to alanine (A) to allow cloning of the TCR expression cassette into the 5' NcoI site of the MSGV1 vector. The expression cassette had the following configuration: 5' NcoI-VDJβ-mCβ-furin / SGSG / P2A-VJα-mCα-SalI3'. The TCR nucleotide sequence was codon-optimized for expression in human T cells using the Genscript codon optimization tool. This example describes the synthesis of a bicistronic vector with a 5' TCRβ→TCRα3' orientation, although the TCRβ→TCRα order can also be reversed. The vector insert sequence was codon-optimized for expression in human tissues.

[0202] Example 9 This example demonstrates the avidity of TCR1 and TCR5 in accordance with an embodiment of the present invention.

[0203] TCR1 and TCR5 were virally transduced into PBLs using the retroviral vectors described in Examples 4 and 8, which were then transduced with various concentrations of RAS. G12V LP or RAS WT The results of flow cytometry assays for 4-1BB and OX40 (% 4-1BB+ / OX40+) expression and ELISPOT measurements for IFN-γ secretion (number of spots per 3e4 cells) are shown in Figures 7A-7G.

[0204] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0205] In connection with the description of the present invention (particularly in connection with the claims which follow), use of the terms "a," "an," "the," and "at least one," and similar referents, should be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise specified herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise specified. The recitation of ranges of values ​​herein, unless otherwise specified herein, is intended to serve merely as a shorthand method for individually referencing each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0206] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. An isolated or purified T cell receptor (TCR), (a) an α chain complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, an α chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, an α chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a β chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a β chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a β chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (b) an α chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, an α chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, an α chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a β chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a β chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a β chain CDR3 comprising the amino acid sequence of SEQ ID NO: 36 Including, have antigen specificity for a variant human RAS amino acid sequence presented by human leukocyte antigen (HLA) class II molecules, and the mutant human RAS amino acid sequence is an amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS); the mutant human RAS amino acid sequence is SEQ ID NO: 30; A TCR, wherein the HLA class II molecule is HLA-DPB1*03:

01.

2. (i) SEQ ID NO: 7, (ii) SEQ ID NO: 129; (iii) SEQ ID NO: 8, (iv) SEQ ID NO: 63, (v) SEQ ID NO: 130, (vi) SEQ ID NO: 64, (vii) SEQ ID NO: 65, (viii) SEQ ID NO: 66, (ix) SEQ ID NO: 37, (x) SEQ ID NO: 38, (xi) SEQ ID NO: 69, (xii) SEQ ID NO: 70, (xiii) SEQ ID NO: 71, (xiv) SEQ ID NO: 47, (xv) SEQ ID NO: 48, (xvi) SEQ ID NO: 49, (xvii) SEQ ID NO: 50, (xviii) SEQ ID NO: 67, (xix) SEQ ID NO: 68, (xx) SEQ ID NO: 76, (xxi) SEQ ID NO: 72, (xxii) SEQ ID NO: 73, (xxiii) SEQ ID NO: 102, or (xxxix) both (i) and (iii); both (i) and (vi); both (i) and (vii); both (i) and (viii); both (ii) and (iii); both (ii) and (vi); both (ii) and (vii); both (ii) and (viii); both (iii) and (iv); both (iii) and (v); both (iv) and (vi); both (iv) and (vii); both (iv) and (viii); both (v) and both (v) and (vi); both (v) and (vii); both (v) and (viii); both (ix) and (x); both (ix) and (xi); both (ix) and (xii); both (ix) and (xiii); both (xiv) and (xv); both (xvi) and (xvii); both (xviii) and (xix); both (xviii) and (xx); both (xxi) and (xxii); or both (xxi) and (xxiii).

2. The isolated or purified TCR of claim 1, comprising the amino acid sequence:

3. (a) an alpha chain constant region comprising the amino acid sequence of SEQ ID NO: 17, (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain constant region in which X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) a β-chain constant region comprising the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b) 3. The isolated or purified TCR of claim 1 or 2, further comprising:

4. (a) an alpha chain comprising the amino acid sequence of SEQ ID NO: 21, (i) X at position 179 of SEQ ID NO: 21 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 246 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) an alpha chain comprising the amino acid sequence of SEQ ID NO: 131, (i) X at position 180 of SEQ ID NO: 131 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 131 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain in which X at position 247 of SEQ ID NO: 131 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (c) a β-strand comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 198 of SEQ ID NO: 22 is Ser or Cys; (d) an alpha chain comprising the amino acid sequence of SEQ ID NO: 79, (i) X at position 179 of SEQ ID NO: 79 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 79 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 79 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 246 of SEQ ID NO: 79 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (e) an alpha chain comprising the amino acid sequence of SEQ ID NO: 134, (i) X at position 180 of SEQ ID NO: 134 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 134 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 134 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain in which X at position 247 of SEQ ID NO: 134 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (f) a β-strand comprising the amino acid sequence of SEQ ID NO: 80, wherein X at position 198 of SEQ ID NO: 80 is Ser or Cys; (g) a β-strand comprising the amino acid sequence of SEQ ID NO: 85, wherein X at position 187 of SEQ ID NO: 85 is Ser or Cys; (h) a β-strand comprising the amino acid sequence of SEQ ID NO: 88, wherein X at position 187 of SEQ ID NO: 88 is Ser or Cys; (i) an alpha chain comprising the amino acid sequence of SEQ ID NO: 41, (i) X at position 179 of SEQ ID NO: 41 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 41 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 246 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (j) a β-strand comprising the amino acid sequence of SEQ ID NO: 42, wherein X at position 197 of SEQ ID NO: 42 is Ser or Cys; (k) a β-strand comprising the amino acid sequence of SEQ ID NO: 105, wherein X at position 197 of SEQ ID NO: 105 is Ser or Cys; (l) a β-strand comprising the amino acid sequence of SEQ ID NO: 110, wherein X at position 186 of SEQ ID NO: 110 is Ser or Cys; (m) a β-strand comprising the amino acid sequence of SEQ ID NO: 113, wherein X at position 186 of SEQ ID NO: 113 is Ser or Cys; (n) both (a) and (c); both (a) and (g); both (b) and (c); both (b) and (g); both (d) and (f); both (d) and (h); both (e) and (f); or both (e) and (h); (o) both (i) and (j); both (i) and (k); both (i) and (l); or both (i) and (m); (p) an alpha chain comprising the amino acid sequence of SEQ ID NO: 55, (i) X at position 160 of SEQ ID NO: 55 is Thr or Cys; (ii) X at position 224 of SEQ ID NO: 55 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 226 of SEQ ID NO: 55 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 227 of SEQ ID NO: 55 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (q) a β-strand comprising the amino acid sequence of SEQ ID NO: 56, wherein X at position 173 of SEQ ID NO: 56 is Ser or Cys; (r) an alpha chain comprising the amino acid sequence of SEQ ID NO: 93, (i) X at position 159 of SEQ ID NO: 93 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 93 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 93 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 226 of SEQ ID NO: 93 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (s) a β-strand comprising the amino acid sequence of SEQ ID NO: 94, wherein X at position 177 of SEQ ID NO: 94 is Ser or Cys; (t) a β-strand comprising the amino acid sequence of SEQ ID NO: 99, wherein X at position 172 of SEQ ID NO: 99 is Ser or Cys; (u) an alpha chain comprising the amino acid sequence of SEQ ID NO: 57, (i) X at position 159 of SEQ ID NO: 57 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 57 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 57 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 226 of SEQ ID NO: 57 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (v) a β-strand comprising the amino acid sequence of SEQ ID NO: 58, wherein X at position 172 of SEQ ID NO: 58 is Ser or Cys; (w) an alpha chain comprising the amino acid sequence of SEQ ID NO: 116, (i) X at position 158 of SEQ ID NO: 116 is Thr or Cys; (ii) X at position 222 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 224 of SEQ ID NO: 116 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain in which X at position 225 of SEQ ID NO: 116 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (x) a β-strand comprising the amino acid sequence of SEQ ID NO: 117, wherein X at position 176 of SEQ ID NO: 117 is Ser or Cys; (y) a β-strand comprising the amino acid sequence of SEQ ID NO: 122, wherein X at position 171 of SEQ ID NO: 122 is Ser or Cys; (z) both (p) and (q); both (r) and (s); or both (r) and (t); (aa) both (u) and (v); both (w) and (x); or both (w) and (y); (bb) an alpha chain comprising the amino acid sequence of SEQ ID NO: 23; (cc) an alpha chain comprising the amino acid sequence of SEQ ID NO: 133; (dd) a β chain comprising the amino acid sequence of SEQ ID NO: 24; (ee) an alpha chain comprising the amino acid sequence of SEQ ID NO: 83; (ff) an alpha chain comprising the amino acid sequence of SEQ ID NO: 136; (gg) a β chain comprising the amino acid sequence of SEQ ID NO: 84; (hh) a β chain comprising the amino acid sequence of SEQ ID NO: 87; (ii) a β-strand comprising the amino acid sequence of SEQ ID NO: 90; (jj) an alpha chain comprising the amino acid sequence of SEQ ID NO: 77; (kk) an alpha chain comprising the amino acid sequence of SEQ ID NO: 132; (ll) a beta chain comprising the amino acid sequence of SEQ ID NO: 78; (mm) an alpha chain comprising the amino acid sequence of SEQ ID NO: 81; (nn) an alpha chain comprising the amino acid sequence of SEQ ID NO: 135; (oo) a β chain comprising the amino acid sequence of SEQ ID NO: 82; (pp) a β-strand comprising the amino acid sequence of SEQ ID NO: 86; (qq) a β chain comprising the amino acid sequence of SEQ ID NO: 89; (rr) an alpha chain comprising the amino acid sequence of SEQ ID NO: 39; (ss) a β-strand comprising the amino acid sequence of SEQ ID NO: 40; (tt) a β chain comprising the amino acid sequence of SEQ ID NO: 107; (uu) a β chain comprising the amino acid sequence of SEQ ID NO: 112; (vv) a β chain comprising the amino acid sequence of SEQ ID NO: 115; (ww) an alpha chain comprising the amino acid sequence of SEQ ID NO: 103; (xx) a β chain comprising the amino acid sequence of SEQ ID NO: 104; (yy) a β chain comprising the amino acid sequence of SEQ ID NO: 106; (zz) a β chain comprising the amino acid sequence of SEQ ID NO: 111; (aaa) a β chain comprising the amino acid sequence of SEQ ID NO: 114; (bbb) both (bb) and (dd); both (cc) and (dd); both (ee) and (gg); both (ff) and (gg); both (bb) and (hh); both (cc) and (hh); both (ee) and (ii); or both (ff) and (ii); (ccc) both (jj) and (ll); both (kk) and (ll); both (mm) and (oo); both (nn) and (oo); both (jj) and (pp); both (kk) and (pp); both (mm) and (qq); or both (nn) and (qq); (ddd) both (rr) and (ss); both (rr) and (tt); both (rr) and (uu); or both (rr) and (vv); (eee) both (ww) and (xx); both (ww) and (yy); both (ww) and (zz); or both (ww) and (aaa); (fff) an alpha chain comprising the amino acid sequence of SEQ ID NO: 51; (ggg) a β chain comprising the amino acid sequence of SEQ ID NO: 52; (hhh) an alpha chain comprising the amino acid sequence of SEQ ID NO: 97; (iii) a β-strand comprising the amino acid sequence of SEQ ID NO: 98; (jjj) a β chain comprising the amino acid sequence of SEQ ID NO: 101; (kkk) an alpha chain comprising the amino acid sequence of SEQ ID NO: 91; (lll) a β chain comprising the amino acid sequence of SEQ ID NO: 92; (mmm) an alpha chain comprising the amino acid sequence of SEQ ID NO: 95; (nnn) a β chain comprising the amino acid sequence of SEQ ID NO: 96; (ooo) a β chain comprising the amino acid sequence of SEQ ID NO: 100; (ppp) an alpha chain comprising the amino acid sequence of SEQ ID NO: 53; (qqq) a β chain comprising the amino acid sequence of SEQ ID NO: 54; (rrr) an a chain comprising the amino acid sequence of SEQ ID NO: 120; (sss) a β chain comprising the amino acid sequence of SEQ ID NO: 121; (ttt) a β chain comprising the amino acid sequence of SEQ ID NO: 124; (uuu) an alpha chain comprising the amino acid sequence of SEQ ID NO: 108; (vvv) a β chain comprising the amino acid sequence of SEQ ID NO: 109; (www) a chain comprising the amino acid sequence of SEQ ID NO: 118; (xxx) a β chain comprising the amino acid sequence of SEQ ID NO: 119; (yyy) a β chain comprising the amino acid sequence of SEQ ID NO: 123; (zzz) both (fff) and (ggg); both (hhh) and (iii); or both (hhh) and (jjj); (aaaa) both (kkk) and (lll); both (mmm) and (nnn); or both (mmm) and (ooo); (bbbb) both (ppp) and (qqq); both (rrr) and (sss); or both (rrr) and (ttt), or (cccc) both (uuu) and (vvv); both (www) and (xxx); or both (www) and (yyy) The isolated or purified TCR of any one of claims 1 to 3, comprising:

5. 5. An isolated or purified polypeptide comprising a functional portion of a TCR according to any one of claims 1 to 4, wherein said functional portion is (a) a TCR alpha chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a TCR alpha chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a TCR alpha chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a TCR beta chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a TCR beta chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a TCR beta chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (b) a TCR α chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a TCR α chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a TCR α chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a TCR β chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a TCR β chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a TCR β chain CDR3 comprising the amino acid sequence of SEQ ID NO: 36 A polypeptide comprising:

6. The functional moiety is (i) SEQ ID NO: 7, (ii) SEQ ID NO: 129; (iii) SEQ ID NO: 8, (iv) SEQ ID NO: 63, (v) SEQ ID NO: 130, (vi) SEQ ID NO: 64, (vii) SEQ ID NO: 65, (viii) SEQ ID NO: 66, (ix) SEQ ID NO: 37, (x) SEQ ID NO: 38, (xi) SEQ ID NO: 69, (xii) SEQ ID NO: 70, (xiii) SEQ ID NO: 71, (xiv) SEQ ID NO: 47, (xv) SEQ ID NO: 48, (xvi) SEQ ID NO: 49, (xvii) SEQ ID NO: 50, (xviii) SEQ ID NO: 67, (xix) SEQ ID NO: 68, (xx) SEQ ID NO: 76, (xxi) SEQ ID NO: 72, (xxii) SEQ ID NO: 73, (xxiii) SEQ ID NO: 102, or (xxxix) both (i) and (iii); both (i) and (vi); both (i) and (vii); both (i) and (viii); both (ii) and (iii); both (ii) and (vi); both (ii) and (vii); both (ii) and (viii); both (iii) and (iv); both (iii) and (v); both (iv) and (vi); both (iv) and (vii); both (iv) and (viii); both (v) and both (v) and (vi); both (v) and (vii); both (v) and (viii); both (ix) and (x); both (ix) and (xi); both (ix) and (xii); both (ix) and (xiii); both (xiv) and (xv); both (xvi) and (xvii); both (xviii) and (xix); both (xviii) and (xx); both (xxi) and (xxii); or both (xxi) and (xxiii).

6. The isolated or purified polypeptide of claim 5, comprising the amino acid sequence(s) of:

7. (a) the amino acid sequence of SEQ ID NO: 17; (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an amino acid sequence in which X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the amino acid sequence of SEQ ID NO: 18, wherein X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b) 7. The isolated or purified polypeptide of claim 5 or 6, further comprising:

8. (a) an alpha chain comprising the amino acid sequence of SEQ ID NO: 21, (i) X at position 179 of SEQ ID NO: 21 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 246 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (b) an alpha chain comprising the amino acid sequence of SEQ ID NO: 131, (i) X at position 180 of SEQ ID NO: 131 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 131 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain in which X at position 247 of SEQ ID NO: 131 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (c) a β-strand comprising the amino acid sequence of SEQ ID NO: 22, wherein X at position 198 of SEQ ID NO: 22 is Ser or Cys; (d) an alpha chain comprising the amino acid sequence of SEQ ID NO: 79, (i) X at position 179 of SEQ ID NO: 79 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 79 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 79 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 246 of SEQ ID NO: 79 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (e) an alpha chain comprising the amino acid sequence of SEQ ID NO: 134, (i) X at position 180 of SEQ ID NO: 134 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 134 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 134 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain in which X at position 247 of SEQ ID NO: 134 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (f) a β-strand comprising the amino acid sequence of SEQ ID NO: 80, wherein X at position 198 of SEQ ID NO: 80 is Ser or Cys; (g) a β-strand comprising the amino acid sequence of SEQ ID NO: 85, wherein X at position 187 of SEQ ID NO: 85 is Ser or Cys; (h) a β-strand comprising the amino acid sequence of SEQ ID NO: 88, wherein X at position 187 of SEQ ID NO: 88 is Ser or Cys; (i) an alpha chain comprising the amino acid sequence of SEQ ID NO: 41, (i) X at position 179 of SEQ ID NO: 41 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 41 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 246 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (j) a β-strand comprising the amino acid sequence of SEQ ID NO: 42, wherein X at position 197 of SEQ ID NO: 42 is Ser or Cys; (k) a β-strand comprising the amino acid sequence of SEQ ID NO: 105, wherein X at position 197 of SEQ ID NO: 105 is Ser or Cys; (l) a β-strand comprising the amino acid sequence of SEQ ID NO: 110, wherein X at position 186 of SEQ ID NO: 110 is Ser or Cys; (m) a β-strand comprising the amino acid sequence of SEQ ID NO: 113, wherein X at position 186 of SEQ ID NO: 113 is Ser or Cys; (n) both (a) and (c); both (a) and (g); both (b) and (c); both (b) and (g); both (d) and (f); both (d) and (h); both (e) and (f); or both (e) and (h); (o) both (i) and (j); both (i) and (k); both (i) and (l); or both (i) and (m); (p) an alpha chain comprising the amino acid sequence of SEQ ID NO: 55, (i) X at position 160 of SEQ ID NO: 55 is Thr or Cys; (ii) X at position 224 of SEQ ID NO: 55 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 226 of SEQ ID NO: 55 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 227 of SEQ ID NO: 55 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (q) a β-strand comprising the amino acid sequence of SEQ ID NO: 56, wherein X at position 173 of SEQ ID NO: 56 is Ser or Cys; (r) an alpha chain comprising the amino acid sequence of SEQ ID NO: 93, (i) X at position 159 of SEQ ID NO: 93 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 93 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 93 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 226 of SEQ ID NO: 93 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (s) a β-strand comprising the amino acid sequence of SEQ ID NO: 94, wherein X at position 177 of SEQ ID NO: 94 is Ser or Cys; (t) a β-strand comprising the amino acid sequence of SEQ ID NO: 99, wherein X at position 172 of SEQ ID NO: 99 is Ser or Cys; (u) an alpha chain comprising the amino acid sequence of SEQ ID NO: 57, (i) X at position 159 of SEQ ID NO: 57 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 57 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 57 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an α chain in which X at position 226 of SEQ ID NO: 57 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (v) a β-strand comprising the amino acid sequence of SEQ ID NO: 58, wherein X at position 172 of SEQ ID NO: 58 is Ser or Cys; (w) an alpha chain comprising the amino acid sequence of SEQ ID NO: 116, (i) X at position 158 of SEQ ID NO: 116 is Thr or Cys; (ii) X at position 222 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 224 of SEQ ID NO: 116 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) an alpha chain in which X at position 225 of SEQ ID NO: 116 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (x) a β-strand comprising the amino acid sequence of SEQ ID NO: 117, wherein X at position 176 of SEQ ID NO: 117 is Ser or Cys; (y) a β-strand comprising the amino acid sequence of SEQ ID NO: 122, wherein X at position 171 of SEQ ID NO: 122 is Ser or Cys; (z) both (p) and (q); both (r) and (s); or both (r) and (t); (aa) both (u) and (v); both (w) and (x); or both (w) and (y); (bb) an alpha chain comprising the amino acid sequence of SEQ ID NO: 23; (cc) an alpha chain comprising the amino acid sequence of SEQ ID NO: 133; (dd) a β chain comprising the amino acid sequence of SEQ ID NO: 24; (ee) an alpha chain comprising the amino acid sequence of SEQ ID NO: 83; (ff) an alpha chain comprising the amino acid sequence of SEQ ID NO: 136; (gg) a β chain comprising the amino acid sequence of SEQ ID NO: 84; (hh) a β chain comprising the amino acid sequence of SEQ ID NO: 87; (ii) a β-strand comprising the amino acid sequence of SEQ ID NO: 90; (jj) an alpha chain comprising the amino acid sequence of SEQ ID NO: 77; (kk) an alpha chain comprising the amino acid sequence of SEQ ID NO: 132; (ll) a beta chain comprising the amino acid sequence of SEQ ID NO: 78; (mm) an alpha chain comprising the amino acid sequence of SEQ ID NO: 81; (nn) an alpha chain comprising the amino acid sequence of SEQ ID NO: 135; (oo) a β chain comprising the amino acid sequence of SEQ ID NO: 82; (pp) a β-strand comprising the amino acid sequence of SEQ ID NO: 86; (qq) a β chain comprising the amino acid sequence of SEQ ID NO: 89; (rr) an alpha chain comprising the amino acid sequence of SEQ ID NO: 39; (ss) a β-strand comprising the amino acid sequence of SEQ ID NO: 40; (tt) a β chain comprising the amino acid sequence of SEQ ID NO: 107; (uu) a β chain comprising the amino acid sequence of SEQ ID NO: 112; (vv) a β chain comprising the amino acid sequence of SEQ ID NO: 115; (ww) an alpha chain comprising the amino acid sequence of SEQ ID NO: 103; (xx) a β chain comprising the amino acid sequence of SEQ ID NO: 104; (yy) a β chain comprising the amino acid sequence of SEQ ID NO: 106; (zz) a β chain comprising the amino acid sequence of SEQ ID NO: 111; (aaa) a β chain comprising the amino acid sequence of SEQ ID NO: 114; (bbb) both (bb) and (dd); both (cc) and (dd); both (ee) and (gg); both (ff) and (gg); both (bb) and (hh); both (cc) and (hh); both (ee) and (ii); or both (ff) and (ii); (ccc) both (jj) and (ll); both (kk) and (ll); both (mm) and (oo); both (nn) and (oo); both (jj) and (pp); both (kk) and (pp); both (mm) and (qq); or both (nn) and (qq); (ddd) both (rr) and (ss); both (rr) and (tt); both (rr) and (uu); or both (rr) and (vv); (eee) both (ww) and (xx); both (ww) and (yy); both (ww) and (zz); or both (ww) and (aaa); (fff) an alpha chain comprising the amino acid sequence of SEQ ID NO: 51; (ggg) a β chain comprising the amino acid sequence of SEQ ID NO: 52; (hhh) an alpha chain comprising the amino acid sequence of SEQ ID NO: 97; (iii) a β-strand comprising the amino acid sequence of SEQ ID NO: 98; (jjj) a β chain comprising the amino acid sequence of SEQ ID NO: 101; (kkk) an alpha chain comprising the amino acid sequence of SEQ ID NO: 91; (lll) a β chain comprising the amino acid sequence of SEQ ID NO: 92; (mmm) an alpha chain comprising the amino acid sequence of SEQ ID NO: 95; (nnn) a β chain comprising the amino acid sequence of SEQ ID NO: 96; (ooo) a β chain comprising the amino acid sequence of SEQ ID NO: 100; (ppp) an alpha chain comprising the amino acid sequence of SEQ ID NO: 53; (qqq) a β chain comprising the amino acid sequence of SEQ ID NO: 54; (rrr) an a chain comprising the amino acid sequence of SEQ ID NO: 120; (sss) a β chain comprising the amino acid sequence of SEQ ID NO: 121; (ttt) a β chain comprising the amino acid sequence of SEQ ID NO: 124; (uuu) an alpha chain comprising the amino acid sequence of SEQ ID NO: 108; (vvv) a β chain comprising the amino acid sequence of SEQ ID NO: 109; (www) a chain comprising the amino acid sequence of SEQ ID NO: 118; (xxx) a β chain comprising the amino acid sequence of SEQ ID NO: 119; (yyy) a β chain comprising the amino acid sequence of SEQ ID NO: 123; (zzz) both (fff) and (ggg); both (hhh) and (iii); or both (hhh) and (jjj); (aaaa) both (kkk) and (lll); both (mmm) and (nnn); or both (mmm) and (ooo); (bbbb) both (ppp) and (qqq); both (rrr) and (sss); or both (rrr) and (ttt), or (cccc) both (uuu) and (vvv); both (www) and (xxx); or both (www) and (yyy) The isolated or purified polypeptide of any one of claims 5 to 7, comprising:

9. (a) a first polypeptide chain comprising a T cell receptor (TCR) alpha chain complementarity determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a TCR alpha chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a TCR alpha chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a second polypeptide chain comprising a TCR beta chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a TCR beta chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a TCR beta chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; or (b) a first polypeptide chain comprising a TCR alpha chain CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a TCR alpha chain CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a TCR alpha chain CDR3 comprising the amino acid sequence of SEQ ID NO: 33; and a second polypeptide chain comprising a TCR beta chain CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a TCR beta chain CDR2 comprising the amino acid sequence of SEQ ID NO: 35, and a TCR beta chain CDR3 comprising the amino acid sequence of SEQ ID NO:

36.

1. An isolated or purified protein comprising: the protein has antigen specificity for a mutant human RAS amino acid sequence presented by a human leukocyte antigen (HLA) class II molecule; the mutant human RAS amino acid sequence is an amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS); the mutant human RAS amino acid sequence is SEQ ID NO: 30; The protein, wherein the HLA class II molecule is HLA-DPB1*03:

01.

10. (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:7 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:8; (ii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (iii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:8; (iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 8; (v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:7 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:64; (vi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64; (vii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64; (viii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 64; (ix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:7 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:65; (x) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 65; (xi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:65; (xii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 65; (xiii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:7 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66; (xiv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 129 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66; (xv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:63 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:66; (xvi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 130 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 66; (xvii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 38; (xviii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69; (xix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 37 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70; (xx) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:37 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:71; (xxi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:47 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:48; (xxii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:68; (xxiii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:67 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:76; (xxiv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:49 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:50; (xxv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73; or (xxvi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:

102.

10. The isolated or purified protein of claim 9.

11. (a) the first polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 17; (i) X at position 48 of SEQ ID NO: 17 is Thr or Cys; (ii) X at position 112 of SEQ ID NO: 17 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 of SEQ ID NO: 17 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 115 of SEQ ID NO: 17 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the second polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 18, and X at position 57 of SEQ ID NO: 18 is Ser or Cys; or (c) Both (a) and (b) 11. The isolated or purified protein of claim 9 or 10,

12. (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:21; (i) X at position 179 of SEQ ID NO: 21 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 21 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 21 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 21 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met, or Trp; (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 131; (i) X at position 180 of SEQ ID NO: 131 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 131 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 131 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 131 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (c) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:22, and X at position 198 of SEQ ID NO:22 is Ser or Cys; (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 79; (i) X at position 179 of SEQ ID NO: 79 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 79 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 79 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 79 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 134; (i) X at position 180 of SEQ ID NO: 134 is Thr or Cys; (ii) X at position 244 of SEQ ID NO: 134 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 246 of SEQ ID NO: 134 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 247 of SEQ ID NO: 134 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (f) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80, and X at position 198 of SEQ ID NO: 80 is Ser or Cys; (g) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85, and X at position 187 of SEQ ID NO: 85 is Ser or Cys; (h) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88, and X at position 187 of SEQ ID NO: 88 is Ser or Cys; (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 41; (i) X at position 179 of SEQ ID NO: 41 is Thr or Cys; (ii) X at position 243 of SEQ ID NO: 41 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 245 of SEQ ID NO: 41 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 246 of SEQ ID NO: 41 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (j) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 42, and X at position 197 of SEQ ID NO: 42 is Ser or Cys; (k) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 105, and X at position 197 of SEQ ID NO: 105 is Ser or Cys; (l) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110, and X at position 186 of SEQ ID NO: 110 is Ser or Cys; (m) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 113, and X at position 186 of SEQ ID NO: 113 is Ser or Cys; (n) both (a) and (c); both (a) and (g); both (b) and (c); both (b) and (g); both (d) and (f); both (d) and (h); both (e) and (f); or both (e) and (h); (o) both (i) and (j); both (i) and (k); both (i) and (l); or both (i) and (m); (p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:55; (i) X at position 160 of SEQ ID NO: 55 is Thr or Cys; (ii) X at position 224 of SEQ ID NO: 55 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 226 of SEQ ID NO: 55 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 227 of SEQ ID NO: 55 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (q) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:56, and X at position 173 of SEQ ID NO:56 is Ser or Cys; (r) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 93; (i) X at position 159 of SEQ ID NO: 93 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 93 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 93 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 226 of SEQ ID NO: 93 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (s) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:94, and wherein X at position 177 of SEQ ID NO:94 is Ser or Cys; (t) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:99, and X at position 172 of SEQ ID NO:99 is Ser or Cys; (u) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:57; (i) X at position 159 of SEQ ID NO: 57 is Thr or Cys; (ii) X at position 223 of SEQ ID NO: 57 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 225 of SEQ ID NO: 57 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 226 of SEQ ID NO: 57 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (v) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:58, and X at position 172 of SEQ ID NO:58 is Ser or Cys; (w) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 116; (i) X at position 158 of SEQ ID NO: 116 is Thr or Cys; (ii) X at position 222 of SEQ ID NO: 116 is Ser, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 224 of SEQ ID NO: 116 is Met, Ala, Val, Leu, He, Pro, Phe, or Trp; and (iv) X at position 225 of SEQ ID NO: 116 is Gly, Ala, Val, Leu, He, Pro, Phe, Met, or Trp; (x) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117, and X at position 176 of SEQ ID NO: 117 is Ser or Cys; (y) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 122, and X at position 171 of SEQ ID NO: 122 is Ser or Cys; (z) both (p) and (q); both (r) and (s); or both (r) and (t); (aa) both (u) and (v); both (w) and (x); or both (w) and (y); (bb) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 23; (cc) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 133; (dd) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 24; (ee) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 83; (ff) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 136; (gg) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84; (hh) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87; (ii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 90; (jj) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77; (kk) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 132; (ll) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 78; (mm) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 81; (nn) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 135; (oo) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82; (pp) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86; (qq) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 89; (rr) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:39; (ss) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (tt) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107; (uu) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 112; (vv) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115; (ww) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 103; (xx) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 104; (yy) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 106; (zz) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 111; (aaa) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114; (bbb) both (bb) and (dd); both (cc) and (dd); both (ee) and (gg); both (ff) and (gg); both (bb) and (hh); both (cc) and (hh); both (ee) and (ii); or both (ff) and (ii); (ccc) both (jj) and (ll); both (kk) and (ll); both (mm) and (oo); both (nn) and (oo); both (jj) and (pp); both (kk) and (pp); both (mm) and (qq); or both (nn) and (qq); (ddd) both (rr) and (ss); both (rr) and (tt); both (rr) and (uu); or both (rr) and (vv); (eee) both (ww) and (xx); both (ww) and (yy); both (ww) and (zz); or both (ww) and (aaa); (fff) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:51; (ggg) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 52; (hhh) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:97; (iii) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 98; (jjj) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 101; (kkk) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:91; (lll) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 92; (mmm) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:95; (nnn) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 96; (ooo) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 100; (ppp) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:53; (qqq) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:54; (rrr) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120; (sss) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 121; (ttt) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 124; (uuu) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108; (vvv) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109; (www) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118; (xxx) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119; (yyy) the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 123; (zzz) both (fff) and (ggg); both (hhh) and (iii); or both (hhh) and (jjj); (aaaa) both (kkk) and (lll); both (mmm) and (nnn); or both (mmm) and (ooo); (bbbb) both (ppp) and (qqq); both (rrr) and (sss); or both (rrr) and (ttt), or (cccc) both (uuu) and (vvv); both (www) and (xxx); or both (www) and (yyy) The isolated or purified protein according to any one of claims 9 to 11,

13. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 8, or a protein according to any one of claims 9 to 12.

14. A recombinant expression vector comprising the nucleic acid of claim 13.

15. 15. An isolated or purified host cell comprising the nucleic acid of claim 13 or the recombinant expression vector of claim 14.

16. 16. A population of isolated or purified cells comprising the host cell of claim 15.

17. 17. A pharmaceutical composition comprising: (a) a TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 8, or a protein according to any one of claims 9 to 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, or a population of cells according to claim 16; and (b) a pharmaceutically acceptable carrier.

18. A pharmaceutical composition for inducing an immune response against cancer in a mammal, comprising as an active ingredient a TCR according to any one of claims 1 to 4, a polypeptide according to any one of claims 5 to 8, or a protein according to any one of claims 9 to 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, a population of cells according to claim 16, or a pharmaceutical composition according to claim 17.

19. A pharmaceutical composition for treating or preventing cancer in a mammal, comprising as an active ingredient the TCR according to any one of claims 1 to 4, the polypeptide according to any one of claims 5 to 8, or the protein according to any one of claims 9 to 12, the nucleic acid according to claim 13, the recombinant expression vector according to claim 14, the host cell according to claim 15, the population of cells according to claim 16, or the pharmaceutical composition according to claim 17; the cancer expresses a mutant human RAS amino acid sequence in which glycine at position 12 is substituted with valine; the mutant human RAS amino acid sequence is an amino acid sequence of a mutant human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutant human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutant human neuroblastoma rat sarcoma viral oncogene homolog (NRAS); A pharmaceutical composition wherein position 12 is defined by reference to wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively.

20. 20. The pharmaceutical composition of claim 18 or 19, wherein the cancer is pancreatic, colorectal, lung, endometrial, ovarian, or prostate cancer.

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