T cell receptors against ras with g12d, g12v, g13d or q61r mutation

Abstract

Disclosed is an isolated or purified T cell receptor (TCR), wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence with a substitution of (i) glycine at position 13 with aspartic acid or (ii) glutamine at position 61 with arginine. 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 of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.

Description

T CELL RECEPTORS AGAINST RAS WITH G12D, G12V, G13D OR Q61R MUTATIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 673,840, filed July 22, 2024, which is incorporated by reference in its entirety herein.STATEMENT REGARDINGFEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under project number ZIABC010984 by the National Institutes of Health. National Cancer Institute. The Government has certain rights in the invention.INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTEDELECTRONICALLY

[0003] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 189,750 Byte Extensible Markup Language (XML) file named “773357. xml;’ dated July 16, 2025.BACKGROUND OF THE INVENTION

[0004] Some cancers may have very limited treatment options, particularly when the cancer becomes metastatic and unresectable. Despite advances in treatments such as, for example, surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as, for example, pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may be poor. Accordingly, there exists an unmet need for additional treatments for cancer.BRIEF SUMMARY OF THE INVENTION

[0005] An aspect of the invention provides an isolated or purified T-cell receptor (TCR) having antigenic specificity for a mutated human RAS amino acid sequence with a substitution of (i) glycine at position 12 with aspartic acid, (ii) glycine at position 12 with valine, (iii) glycine at position 13 with aspartic acid or (iv) glutamine at position 61 witharginine, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutated human Han ey rat sarcoma viral oncogene homolog (HRAS), or a mutated human Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, wherein positions 12, 13 and 61 are defined by reference to the wild-ty pe human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively, and wherein the TCR comprises the amino acid sequences of: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 1-6, (d) all of SEQ ID NOs: 11-13, (e) all of SEQ ID NOs: 14-16, (f) all of SEQ ID NOs: 11-16, (g) all of SEQ ID NOs: 21-23. (h) all of SEQ ID NOs: 24-26. (i) all of SEQ ID NOs: 21-26, (j) all of SEQ ID NOs: 31-33. (k) all of SEQ ID NOs: 34-36. (1) all of SEQ ID NOs: 31-36, (m) all of SEQ ID NOs: 41-43, (n) all of SEQ ID NOs: 44-46, (o) all of SEQ ID NOs: 41-46, (p) all of SEQ ID NOs: 101-103, (q) all of SEQ ID NOs: 104-106, (r) all of SEQ ID NOs: 101-106, (s) all of SEQ ID NOs: 111-113, (t) all of SEQ ID NOs: 114-116, (u) all of SEQ ID NOs: 111- 116, (v) all of SEQ ID NOs: 121-123, (w) all of SEQ ID NOs: 124-126, (x) all of SEQ ID NOs: 121-126, (y) all of SEQ ID NOs: 131-133, (z) all of SEQ ID NOs: 134-136, (aa) all of SEQ ID NOs: 131-136, (bb) all of SEQ ID NOs: 141-143, (cc) all of SEQ ID NOs: 144-146, (dd) all of SEQ ID NOs: 141-146, (ee) all of SEQ ID NOs: 151-153, (fl) all of SEQ ID NOs: 154-156, or (gg) all of SEQ ID NOs: 151-156.

[0006] Another aspect of the invention provides an isolated or punfied polypeptide comprising a functional portion of the inventive TCR, wherein the functional portion comprises the amino acid sequences of: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 1-6, (d) all of SEQ ID NOs: 11-13, (e) all of SEQ ID NOs: 14- 16. (1) all of SEQ ID NOs: 11-16, (g) all of SEQ ID NOs: 21-23, (h) all of SEQ ID NOs: 24- 26, (i) all of SEQ ID NOs: 21-26, (j) all of SEQ ID NOs: 31 -33, (k) all of SEQ ID NOs: 34- 36, (1) all of SEQ ID NOs: 31-36, (m) all of SEQ ID NOs: 41-43, (n) all of SEQ ID NOs: 44- 46, (o) all of SEQ ID NOs: 41-46, (p) all of SEQ ID NOs: 101-103. (q) all of SEQ ID NOs: 104-106, (r) all of SEQ ID NOs: 101-106, (s) all of SEQ ID NOs: 111-113. (t) all of SEQ ID NOs: 1 14-116, (u) all of SEQ ID NOs: 11 1-116, (v) all of SEQ ID NOs: 121-123, (w) all of SEQ ID NOs: 124-126, (x) all of SEQ ID NOs: 121-126, (y) all of SEQ ID NOs: 131-133, (z) all of SEQ ID NOs: 134-136, (aa) all of SEQ ID NOs: 131-136. (bb) all of SEQ ID NOs: 141-143, (cc) all of SEQ ID NOs: 144-146, (dd) all of SEQ ID NOs: 141-146, (ee) all of SEQ ID NOs: 151-153, (ff) all of SEQ ID NOs: 154-156. or (gg) all of SEQ ID NOs: 151-156.

[0007] Still another aspect of the invention provides an isolated or purified protein, comprising: (a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 1-3 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 4-6; (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 11-13 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 14-16; (c) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 21- 23 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 24- 26; (d) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 31-33 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 34-36; (e) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 41-43 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 44-46; (1) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 101-103 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 104-106; (g) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 111-113 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 114- 116; (h) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 121 - 123 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 124-126; (i) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 131-133 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 134-136; (j) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 141-143 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 144-146; or (k) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 151-153 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 154-156.

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

[0009] An aspect of the invention provides 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 sequence, respectively, encode the amino sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 17 and 18; 18 and 17; 19 and 20; 20 and 19; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 37 and 38; 38 and 37; 39 and 40; 40 and 39; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 69 and 70; 70 and 69; 71 and 72; 72 and 71;73 and 74; 74 and 73; 75 and 76; 76 and 75; 77 and 78; 78 and 77; 79 and 80; 80 and 79; 81 and 82; 82 and 81 ; 83 and 84; 84 and 83; 85 and 86; 86 and 85; 87 and 88; 88 and 87; 107 and 108; 108 and 107; 109 and 110; 110 and 109; 117 and 118; 118 and 117; 119 and 120; 120 and 119; 127 and 128; 128 and 127; 129 and 130; 130 and 129; 137 and 138; 138 and 137; 139 and 140; 140 and 139; 147 and 148; 148 and 147; 149 and 150; 150 and 149; 157 and 158; 158 and 157; 159 and 160; 160 and 159; 161 and 162; 162 and 161; 163 and 164; 164 and 163; 165 and 166; 166 and 165; 167 and 168; 168 and 167; 169 and 170; 170 and 169; 171 and 172; 172 and 171; 173 and 174; 174 and 173; 175 and 176; 176 and 175; 177 and 178; 178 and 177; 179 and 180; 180 and 179; 181 and 182; 182 and 181; 183 and 184; or 184 and 183.

[0010] Another aspect of the invention provides a method of producing a host cell expressing a TCR that has antigenic specificity for the peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89), MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91), MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95), VVGAVGVGK (SEQ ID NO: 97), or VVVGAVGVGK (SEQ ID NO: 99), the method comprising contacting a cell with the inventive recombinant expression vector under conditions that allow introduction of the vector into the cell.

[0011] Still another aspect of the invention provides a method of producing the inventive TCR, polypeptide, or protein, the method comprising culturing the inventive host cell or the population of host cells so that the inventive TCR, polypeptide, or protein is produced.

[0012] Another aspect of the invention provides a method of detecting the presence of cancer in mammal, the method comprising: (a) contacting a sample comprising cells of the cancer with the inventive TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population, or pharmaceutical composition, thereby forming a complex; and (b) detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal.

[0013] Another aspect of the invention provides a method of inducing an immune response against cancer in a mammal, the method comprising administering to the mammal the inventive TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population, or pharmaceutical composition, in an amount effective to induce the immune response against cancer in the mammal.

[0014] Another aspect of the invention provides a method of treating or preventing cancer in a mammal, the method comprising administering to the mammal the inventive TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population, or pharmaceutical composition in an amount effective to treat or prevent cancer in the mammal.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0015] Figure 1 is a graph showing the percentage of CD3+, murine TCR (mTCR) positive cells expressing 4-1BB following co-culture of target cells with effector cells. Effector cells were healthy donor PBL transduced with a retroviral vector encoding the 4589 NEO4 TCR 1. Target cells were DCs pulsed with the indicated concentrations of G12D peptide or the corresponding WT peptide.

[0016] Figure 2 shows the proportion of TCR-transduced, PE- or APC-tetramer stained cells measured by flow cytometry. The cells were independently transduced with a retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 and stained with fluorescent HLA-A* 11 :01 RAS G12V tetramers containing the predicted 9-mer neoepitope having the amino acid sequence of VVGAVGVGK (SEQ ID NO: 97) or the predicted 10-mer neoepitope having the amino acid sequence of VVVGAVGV GK (SEQ ID NO: 99). The cells were pre-gated on CD8+ mTCR+ cells.

[0017] Figures 3A-3E are graphs showing the percentage of CD8+, murine TCR (mTCR) positive cells expressing 4- IBB following co-culture of target cells with effector cells.Effector cells were healthy donor PBL transduced with a retroviral vector encoding the 4424 TCR 11 (A), 4424 TCR 12 (B), 4424 TCR 13B (C), 4424 TCR 17 (D), or 4424 TCR 18 (E). Target cells were allogenic, HLA-matched EBV transformed B cells pulsed with the indicated concentrations of G12V 9-mer peptide or the corresponding WT peptide.

[0018] Figures 4A-4E are graphs showing the percentage of CD8+. munne TCR (mTCR) positive cells expressing 4-1BB following co-culture of target cells with effector cells.Effector cells were healthy donor PBL transduced with a retroviral vector encoding the 4424 TCR 11 (A), 4424 TCR 12 (B), 4424 TCR 13B (C). 4424 TCR 17 (D), or 4424 TCR 18 (E). Target cells were allogenic, HLA-matched EBV transformed B cells pulsed with the indicated concentrations of G12V 10-mer peptide or the corresponding WT peptide.

[0019] Figure 5 is a graph showing IFN-y secretion (spots / 2e4 cells) measured by ELISPOT assay following co-culture of effector cells with target cells. Effector cells were T cells transduced with a retroviral vector encoding 4589 NEO4 TCR 1. Target cells were COScells transfected with the indicated HLA Class II heterodimers and pulsed with the G12D peptide MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95).

[0020] Figures 6A-6F are graphs showing the percentage of effector cells that upregulated 4- IBB expression following co-culture of effector cells with target cells. Effector cells were CD4+ or CD8+ PBL independently transduced with a retroviral vector encoding 4424 TCR 11 (6D), 4424 TCR 12 (6C), 4424 TCR 13B (6 A), 4424 TCR 17 (6F), or 4424 TCR 18 (6E). Target cells were B cells pulsed with a 9-mer G12V peptide or the corresponding WT peptide at the indicated concentrations. PBL transduced with 4148 TCR 2 (6B) served as control effector cells.

[0021] Figure 7 is a graph showing the percentage of effector cells that upregulated 4- 1BB expression following co-culture of effector cells w ith target cells. Effector cells were CD3+, CD4+ or CD8+ PBL independently transduced with a retroviral vector encoding 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18. Target cells were a human tumor line engineered to express HLA A* 11 :01. CD3+, CD4+ or CD8+ PBL transduced with 4148 TCR 2 served as control effector cells.

[0022] Figures 8A-8B are graphs showing the percentage of effector cells that upregulated 4- IBB expression following co-culture of effector cells with target cells. Effector cells were CD8+ (8A) or CD4+ (8B) PBL independently transduced with a retroviral vector encoding 4424 TCR 11, 4424 TCR 12. 4424 TCR 13B, 4424 TCR 17. or 4424 TCR 18. Target cells were a human tumor organoid (4626) pretreated with interferon gamma (4626org+ifny) or without any pre-treatment (4626org). Human tumor organoid (4424), with HLA-A* 11 :01 loss of heterozygosity (LOH), pretreated with interferon gamma (4424org+ifny) or without any pre-treatment (4424org) served as negative control target cells. CD4+ or CD8+ PBL transduced with 4148 TCR 2 served as control effector cells.

[0023] Figure 9 is a schematic illustrating the methods used in the experiment described in Example 35. “DO” is Day 0. “D14” is Day 14. “D21+” is Day 21 and subsequent days.

[0024] Figure 10 is a graph showing the tumor size (mm2) measured in tumor-bearing mice on the days following treatment with adoptive cell transfer (ACT) of CD8-enriched cells independently transduced with a retroviral vector encoding 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18. CD8+-enriched cells transduced with an empty vector (Mock) or 4148 TCR 2 served as controls. Statistical comparisons were by Two-way ANOVA Tukey's multiple corrections.

[0025] Figures 11 A-l IB are graphs showing the percentage of effector cells that upregulated 4-1 BB expression following co-culture of effector cells with target cells. Effector cells were CD8+ (11 A) or CD4+ (1 IB) PBL independently transduced with a retroviral vector encoding 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18. Target cells were a human tumor organoid (8045) pretreated with interferon gamma (8045org+ifhy) or without any pre-treatment (8045org) or patient 8045-derived xenograft (8045 PDX). Human tumor organoid (4447), with an irrelevant HLA-mutation combination and pretreated with interferon gamma (4447org+ifny) or without any pretreatment (4447org), served as negative control target cells. CD4+ or CD8+ PBL transduced with 4148 TCR 2 served as control effector cells.

[0026] Figure 12 is a graph showing the percentage of effector cells that expressed 4- IBB following co-culture of effector cells with target cells. Effector cells were PBL transduced with the 4342 TCR 8. Target cells were APCs from Patient 4508 or Patient 4487 pulsed with 25-mer peptide containing the KRAS Q61R neoepitope or the corresponding WT peptide at one of the indicated concentrations.

[0027] Figure 13 is a graph showing the relative CD8 coreceptor dependence of 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, 4424 TCR 18, and 4148 TCR 2 bytetramer and CD8 / CD4 staining.DETAILED DESCRIPTION OF THE INVENTION

[0028] RAS family proteins belong to the large family of small GTPases. Without being bound to a particular theory or mechanism, it is believed that, when mutated, RAS proteins may be involved in signal transduction early in the oncogenesis of many human cancers. A single amino acid substitution may activate the protein. The mutated RAS protein product may be constitutively activated. Mutated RAS proteins may be expressed in any of a variety of human cancers such as, for example, pancreatic (e.g., pancreatic carcinoma), colorectal, lung (e.g., lung adenocarcinoma), endometrial, ovarian (e.g., epithelial ovarian cancer), and prostate cancers. The human RAS family proteins include KRAS, HRAS, and NRAS.

[0029] KRAS is also referred to as GTPase KRas. V-Ki-Ras2 Kirsten rat sarcoma viral oncogene, or KRAS2. There are two transcript variants of KRAS: KRAS variant A and KRAS variant B. Wild-type (WT) KRAS variant A has the amino acid sequence of SEQ ID NO: 51. WT KRAS variant B has the amino acid sequence of SEQ ID NO: 52. Hereinafter, references to “KRAS” (mutated or unmutated (WT)) refer to both variant A and variant B,unless specified otherwise. When activated, mutated KRAS binds to guanosine-5'- triphosphate (GTP) and converts GTP to guanosine 5'-diphosphate (GDP).

[0030] HRAS is another member of the RAS protein family. HRAS is also referred to 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: 53.

[0031] NRAS is still another member of the RAS protein family. NRAS is also referred to as GTPase NRas, V-Ras Neuroblastoma RAS Viral Oncogene Homolog, or NRAS1. WT NRAS has the amino acid sequence of SEQ ID NO: 54.

[0032] An aspect of the invention provides an isolated or purified TCR, wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence with a substitution of (i) glycine at position 12 with aspartic acid, (ii) glycine at position 12 with valine, (iii) glycine at position 13 with aspartic acid or (iv) glutamine at position 61 with arginine, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, a mutated human HRAS, or a mutated human NRAS amino acid sequence, and wherein positions 12, 13 and 61 are defined by reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. Hereinafter, references to a “TCR'’ also refer to functional portions and functional variants of the TCR, unless specified otherwise.

[0033] The mutated human RAS amino acid sequence may be a mutated human KRAS amino acid sequence, a mutated human HRAS amino acid sequence, or a mutated human NRAS amino acid sequence. The amino acid sequences of WT human KRAS, NRAS, and HRAS protein each have a length of 188 or 189 amino acid residues and have a high degree of identity to one another. For example, the amino acid sequence of the WT human NRAS protein is 86.8% identical to that of the WT human KRAS protein. Amino acid residues 1 -86 of the WT human NRAS protein and the WT human KRAS protein are 100% identical. The amino acid sequence of the WT human HRAS protein is 86.3% identical to that of the WT human KRAS protein. Amino acid residues 1-94 of the WT human HRAS protein and the WT human KRAS protein are 100% identical. Hereinafter, references to “RAS” (mutated or unmutated (WT)) collectively refer to KRAS, HRAS, and NRAS, unless specified otherwise.

[0034] In an aspect of the invention, the mutated human RAS amino acid sequence comprises a human RAS amino acid sequence with a substitution of glycine at position 12 with aspartic acid, wherein position 12 is defined by reference to the corresponding WT RAS protein. In another aspect of the invention, the mutated human RAS amino acid sequencecomprises a human RAS amino acid sequence with a substitution of glycine at position 12 with valine, wherein position 12 is defined by reference to the corresponding WT RAS protein. In another aspect of the invention, the mutated human RAS amino acid sequence comprises a human RAS amino acid sequence with a substitution of glycine at position 13 with aspartic acid, wherein position 13 is defined by reference to the corresponding WT RAS protein. In another aspect of the invention, the mutated human RAS amino acid sequence comprises a human RAS amino acid sequence with a substitution of glutamine at position 61 with arginine, wherein position 61 is defined by reference to the corresponding WT RAS protein.

[0035] The WT RAS protein may be any one of WT KRAS protein (SEQ ID NO: 51 or 52), WT HRAS protein (SEQ ID NO: 53), or WT NRAS protein (SEQ ID NO: 54) 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 100% identical. Accordingly, the amino acid residue at position 12 of each of WT KRAS, WT HRAS, and WT NRAS protein is the same, namely, glycine; the amino acid residue at position 13 of each of WT KRAS, WT HRAS, and WT NRAS protein is the same, namely, glycine; and the amino acid residue at position 61 of each of WT KRAS, WT HRAS, and WT NRAS protein is the same, namely, glutamine.

[0036] In an aspect of the invention, the mutated human RAS amino acid sequence has a substitution of glycine at position 12 with aspartic acid. In this regard, aspects of the invention provide TCRs with antigenic specificity for any human RAS protein, polypeptide or peptide amino acid sequence with a G12D mutation.

[0037] In an aspect of the invention, the mutated human RAS amino acid sequence has a substitution of glycine at position 12 with valine. In this regard, aspects of the invention provide TCRs with antigenic specificity for any human RAS protein, polypeptide or peptide amino acid sequence with a G12V mutation.

[0038] In an aspect of the invention, the mutated human RAS amino acid sequence has a substitution of glycine at position 13 with aspartic acid. In this regard, aspects of the invention provide TCRs with antigenic specificity for any human RAS protein, polypeptide or peptide amino acid sequence with a G13D mutation.

[0039] In an aspect of the invention, the mutated human RAS amino acid sequence has a substitution of glutamine at position 61 with arginine. In this regard, aspects of the inventionprovide TCRs with antigenic specificity for any human RAS protein, polypeptide or peptide amino acid sequence with a Q61 R mutation.

[0040] Mutations and substitutions of RAS are defined herein by reference to the amino acid sequence of the corresponding WT RAS protein. Thus, mutations and substitutions of RAS are described herein by reference to the amino acid residue present at a particular position in WT RAS protein (namely, position 12, 13 or 61), followed by the position number, followed by the amino acid residue with which that residue has been replaced in the particular mutation or substitution under discussion. A RAS amino acid sequence (e.g., a RAS peptide) may comprise fewer than all of the amino acid residues of the full-length, WT RAS protein. Accordingly, positions 12, 13 and 61 are defined herein by reference to the WT full-length RAS protein (namely, any one of SEQ ID NOs: 51-54) with the understanding that the actual position of the corresponding residue in a particular example of a RAS amino acid sequence may be different.

[0041] When the positions are as defined by any one of SEQ ID NOs: 51-54. the term “G12” refers to the glycine normally present at position 12 of any one of SEQ ID NOs: 51- 54, “G12D” indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 51-54 is replaced by aspartic acid, and “G12V” indicates that the glycine normally present at position 12 of any one of SEQ ID NOs: 51-54 is replaced by valine. For example, when a particular example of a RAS amino acid sequence is, e.g..TEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 94) (an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 2 to 24 of SEQ ID NO: 51), “G12D” refers to a substitution of the underlined glycine in SEQ ID NO: 94 with aspartic acid, even though the actual position of the underlined glycine in SEQ ID NO: 94 is 11. Human RAS amino acid sequences with the G12D mutation are hereinafter referred to as '‘G12D RAS,” “RAS G12D,” or “G12D.” Human RAS amino acid sequences with the G12V mutation are hereinafter referred to as “G12V RAS.” “RAS G12V,” or “G12V.”

[0042] When the positions are as defined by any one of SEQ ID NOs: 51-54. the term “G13” refers to the glycine normally present at position 13 of any one of SEQ ID NOs: 51- 54, and “G13D” indicates that the glycine normally present at position 13 of any one of SEQ ID NOs: 51-54 is replaced by aspartic acid. For example, when a particular example of a RAS ammo acid sequence is, e g., TEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 94) (an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 2 to 24 of SEQ ID NO: 51), “G13D” refers to a substitution of the underlined glycine in SEQ ID NO:94 with aspartic acid, even though the actual position of the underlined glycine in SEQ ID NO: 94 is 12. Human RAS amino acid sequences with the G13D mutation are hereinafter referred to as “G13D RAS,” “RAS G13D,” or “G13D.”

[0043] When the positions are as defined by any one of SEQ ID NOs: 51-54, the term “Q61” refers to the glutamine normally present at position 61 of any one of SEQ ID NOs: 51- 54, and “Q61R” indicates that the glutamine normally present at position 61 of any one of SEQ ID NOs: 51-54 is replaced by arginine. For example, when a particular example of a RAS amino acid sequence is, e g., ETCLLDILDTAGQEEYSAMRDQYMR (SEQ ID NO: 90) (an exemplary WT KRAS peptide corresponding to contiguous amino acid residues 49 to 73 of SEQ ID NO: 51), “Q61R” refers to a substitution of the underlined glutamine in SEQ ID NO: 90 with arginine, even though the actual position of the underlined glycine in SEQ ID NO: 90 is 13. Human RAS amino acid sequences with the Q61R mutation are hereinafter referred to as “Q61R RAS,” “RAS Q61R,” or “Q61R.”

[0044] Examples of full-length RAS proteins with the G13D mutation are set forth in Table 1 below.TABLE 1

[0045] Examples of full-length RAS proteins with the Q61R mutation are set forth in Table 2A below.TABLE 2A

[0046] Examples of full-length RAS proteins with the G12D or G12V mutation are set forth in Table 2B below.TABLE 2B

[0047] In an aspect of the invention, the TCR has antigenic specificity for a RAS peptide with the G12D, G13D or Q61R mutation described above, wherein the G12D RAS, G13D RAS or Q61R RAS peptide has any length. In an aspect of the invention, the G12D RAS, G13D RAS or Q61R RAS peptide has any length suitable for binding to any of the HLA Class II molecules described herein. For example, the TCR may have antigenic specificity' for a RAS peptide with the G12D, G13D or Q61R mutation, the RAS peptide having a length of 11 to 30 amino acid residues, 12 to 24 amino acid residues, or 18 to 20 amino acid residues. The G12D RAS. G13D RAS or Q61R RAS peptide may comprise any contiguous amino acid residues of mutated RAS protein which include the G12D, G13D or Q61R mutation, respectively. In an aspect of the invention, the TCR may have antigenic specificity for a RAS peptide with the G12D, G13D or Q61R mutation, the mutated RAS peptide having a length of 30 amino acid residues, 29 amino acid residues, 28 amino acid residues, 27 amino acid residues, 26 amino acid residues, 25 amino acid residues, 24 amino acid residues, 23 amino acid residues, 22 amino acid residues, 21 amino acid residues, 20 amino acid residues, 19 amino acid residues, 18 amino acid residues, 17 amino acid residues, 16 amino acid residues, 15 amino acid residues, 14 amino acid residues, 13 amino acid residues, 12 amino acid residues, 11 amino acid residues, or a range of any two of the foregoing values.

[0048] An example of a specific peptide with the G12D mutation, which may be recognized by the inventive TCRs, is MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95). In an aspect of the invention, the TCR has antigenic specificity for the mutated humanRAS amino acid sequence of SEQ ID NO: 95. In an aspect of the invention, the TCR does not have antigenic specificity for the wild-type human RAS amino acid sequence of MTEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 96).

[0049] An example of a specific peptide with the G13D mutation, which may be recognized by the inventive TCRs, is MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91). In an aspect of the invention, the TCR has antigenic specificity for the mutated human RAS amino acid sequence of SEQ ID NO: 91. In an aspect of the invention, the TCR does not have antigenic specificity' for the wild-type human RAS amino acid sequence of MTEYKLVVVGAGGVGKSALTIQLIQ (SEQ ID NO: 92).

[0050] An example of a specific peptide with the Q61R mutation, which may be recognized by the inventive TCRs, is ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89). In an aspect of the invention, the TCR has antigenic specificity for the mutated human RAS amino acid sequence of SEQ ID NO: 89. In an aspect of the invention, the TCR does not have antigenic specificity for the wild-type human RAS amino acid sequence of ETCLLDILDTAGQEEYSAMRDQYMR (SEQ ID NO: 90).

[0051] In an aspect of the invention, the inventive TCRs are able to recognize G12DRAS, G13D RAS or Q61R RAS presented by an HLA Class II molecule. In this regard, the TCR may elicit an immune response upon binding to G12D RAS, G13D RAS or Q61R RAS within the context of an HLA Class II molecule. The inventive TCRs are able to recognize G12D RAS, G13D RAS or Q61R RAS that is presented by an HLA Class II molecule and may bind to the HLA Class II molecule in addition to G12D RAS, G13D RAS or Q61R RAS, respectively.

[0052] In an aspect of the invention, the HLA Class II molecule is an HLA-DQ heterodimer. The HLA-DQ heterodimer is a cell surface receptor including an a chain and a P chain. The HLA-DQ a chain is encoded by the HLA-DQA1 gene. HLA-DQA1 alleles may include DQAl*01:01, DQAl*01:02, DQA1 *01:03, DQAl*01:04, DQAl*02:01, DQAl*03:01, DQAl*03:02, DQA1*O3:O3, DQAl*04:01, DQAI *05:01, DQA1*O5:O5, and DQAl*06:01. The HLA-DQ chain is encoded by the HLA-DQB1 gene. HLA-DQB1 alleles may include HLA-DQB 1*02:01, HLA-DQBl*02:02, HLA-DQBl*02:03, HLA- DQBl*03:01, HLA-DQB 1*03: 02, HLA-DQBl*03:03, HLA-DQB 1*03: 04, HLA- DQBl*03:05, HLA-DQB 1*04: 01, HLA-DQBl*04:02, HLA-DQB 1*05:01, HLA- DQBl*05:02, HLA-DQB 1*05: 03, HLA-DQB 1*05: 04. HLA-DQBl*06:01, HLA- DQBl*06:02, HLA-DQB 1*06: 03, HLA-DQB 1*06: 04, HLA-DQBl*06:05, and HLA-DQBl*06:09. In an aspect of the invention, the HLA Class II molecule is an HLA- DQA1 :HLA-DQB1 heterodimer. In an especially preferred aspect, the HLA Class II molecule is expressed by the HLA-DQAl*01 :01:HLA-DQBl*05:01 allele, the HLA- DQA1*O5:O5:HLA-DQB1*O3:O1 allele. the HLA-DQAl*05:01:HLA-DQBl*03:01 allele, the HLA-DQAl*01:01:HLA-DQBl*03:01 allele, the HLA-DQA1 *01 :02:HLA- DQBl*05:02 allele, or the HLA-DQAl*01:02:HLA-DQBl*02:02 allele (namely, an HLA- DQA1 *01 :01:HLA-DQBl *05:01 heterodimer, an HLA-DQAl*05:05:HLA-DQBl*03:01 heterodimer, an HLA-DQAl*05:01:HLA-DQBl*03:01 heterodimer, an HLA- DQA1 *01 : 01 :HLA-DQB 1*03:01 heterodimer, an HLA-DQA1 *01 : 02:HLA-DQB 1*05: 02 heterodimer, or an HLA-DQAl*01 :02:HLA-DQBl*02:02 heterodimer).

[0053] In an aspect of the invention, the HLA Class II molecule is an HLA-DR heterodimer. The HLA-DR heterodimer is a cell surface receptor including an a chain and a P chain. The HLA-DR a chain is encoded by the HLA-DRA gene. The HLA-DR chain is encoded by the HLA-DRB 1 gene, the HLA-DRB3 gene, HLA-DRB4 gene, or the HLA- DRB5 gene. Examples of molecules encoded by the HLA-DRB 1 gene may include, but are not limited to, HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA- DR?, HLA-DR8. HLA-DR9, HLA-DR10. HLA-DR11, HLA-DR12, HLA-DR 13. HLA- DR14, HLA-DR15, HLA-DR16, and HLA-DR17. The HLA-DRB3 gene encodes HLA- DR52. The HLA-DRB4 gene encodes HLA-DR53. The HLA-DRB5 gene encodes HLA- DR51. In an aspect of the invention, the HLA Class II molecule comprises a HLA-DR a chain in combination with a HLA-DR P chain encoded by the HLA-DRB 1 gene. In an especially preferred aspect, the HLA Class II molecule is an HLA-DRALHLA-DRB1 heterodimer (namely, expressed by the HLA-DRA l*01:HLA-DRB 1*04 alleles). In an especially preferred aspect, the HLA Class II molecule is an HLA-DRA1 *01 :01 :HLA- DRBl*04:05 heterodimer.

[0054] In an aspect of the invention, the TCR has antigenic specificity for a RAS peptide with the G12V mutation described above, wherein the G12V RAS peptide has any length. In an aspect of the invention, the G12V RAS peptide has any length suitable for binding to any of the HLA Class I molecules described herein. For example, the TCR may have antigenic specificity7for a RAS peptide with the G12V mutation, the G12V RAS peptide having a length of about 9 to about 10 amino acid residues. The G12V RAS peptide may comprise any contiguous amino acid residues of mutated RAS protein which include the GI2V mutation. Examples of specific peptides with the G12V mutation, which may be recognizedby the inventive TCRs, is VVGAVGVGK (SEQ ID NO: 97) or VVVGAVGVGK (SEQ ID NO: 99). In an aspect of the invention, the TCR has antigenic specificity for the mutated human RAS amino acid sequence of SEQ ID NO: 97 or 99. In an aspect of the invention, the TCR does not have antigenic specificity for the wild-type human RAS amino acid sequence of VVGAGGVGK (SEQ ID NO: 98) or VVVGAGGVGK (SEQ ID NO: 100).

[0055] In an aspect of the invention, the inventive TCRs are able to recognize G12V RAS presented by an HLA Class I molecule. In this regard, the TCR may elicit an immune response upon binding to G12V RAS presented by an HLA Class I molecule. The inventive TCRs may bind to the HLA Class I molecule in addition to G12V RAS.

[0056] In an aspect of the invention, the HLA Class I molecule is an HLA-A molecule. The HLA-A molecule is a heterodimer of an a chain and P2 microglobulin. The HLA-A a chain may be encoded by an HLA-A gene. [ 2 microglobulin binds non-covalently to the alphal, alpha2 and alpha3 domains of the alpha chain to build the HLA-A complex. The HLA-A molecule may be any HLA-A molecule. In an aspect of the invention, the HLA Class I molecule is an HLA-A 11 molecule. The HLA-A11 molecule may be any HLA-A11 molecule. Examples of HLA-A11 molecules may include, but are not limited to, those encoded by the HLA-A* 11 :01, HLA-A*l l:02, HLA-A*ll:03 allele, or HLA-A* 11:04. Preferably, the HLA Class I molecule is encoded by the HLA-A* 11 :01 allele.

[0057] The TCRs of the invention may provide any one or more of a variety of advantages, including when expressed by cells used for adoptive cell transfer. G12D RAS, G12V RAS, G13D RAS and Q61R RAS are expressed by cancer cells and are not expressed by normal, noncancerous cells. Without being bound to a particular theory or mechanism, it is believed that the inventive TCRs advantageously target the destruction of cancer cells while minimizing or eliminating the destruction of normal, non-cancerous cells, thereby reducing, for example, by minimizing or eliminating, toxicity. Moreover, because the G12D, G12V, G13D and Q61R mutations are likely to occur in the early stages of tumorigenesis, the G12D RAS, G12V RAS, G13D RAS or Q61R RAS mutation, respectively, may be expressed on substantially all of a patient’s cancer cells. The inventive TCRs may, advantageously, successfully treat or prevent G12D RAS-positive, G12V RAS-positive, G13D RAS-positive or Q61R RAS-positive cancers that do not respond to other types of treatment such as, for example, chemotherapy, surgery, or radiation. Additionally, the inventive TCRs may provide highly avid recognition of G12D RAS, G12V RAS. GI3D RAS or Q61R RAS, which may provide the ability to recognize unmanipulated tumor cells (e.g., tumor cells that have notbeen treated with interferon (IFN)-y. transfected with a vector encoding (i) G12D, G12V, G13D or Q61R RAS and (ii) the applicable HLA molecule, pulsed with a G12D RAS, G12V RAS, G13D RAS or Q61R peptide, or a combination thereof). KRAS is a common protooncogene in cancers including, for example, lung adenomcarcinoma, mucinous adenocarcinoma, colorectal carcinoma, and pancreatic adenocarcinoma. For example, a KRAS mutation is found in nearly 90% of pancreatic cancers, 30-40% of colon cancers, and 15-20% of lung cancers. KRAS mutations are found in about 30% of all cancer patients. The G12V RAS mutation is found in about 27% and about 8% of patients with pancreatic and colorectal cancers, respectively. The G12D RAS mutation is found in about 40% and about 12% of patients with pancreatic and colorectal cancers, respectively. The G13D RAS mutation is found in about 19% of cancer patients with a KRAS mutation. NRAS is a protooncogene associated with multiple cancers such as rectal cancer, follicular thyroid cancer, cutaneous melanoma, and leukemia. The most common NRAS mutation occurs at position 61 as either a Q61R or Q61K mutation and is detected in about 20% of melanomas. The inventive TCRs may increase the number of immunotherapy-eligible cancer patients to include those patients that express the HLA-DQA1*O1 :O1 :HLA-DQB1*O5:O1, HLA- DQAl*05:05:HLA-DQBl*03:01, HLA-DQAl*05:01 :HLA-DQBl*03:01, HLA- DRA1 *01 :01 :HLA-DRB 1*04:05, or HLA-A* 11 :01 alleles who may not be eligible for immunotherapy using TCRs that recognize RAS presented by other MHC molecules.Moreover, the inventive TCRs, polypeptides and proteins comprise human CDR and variable region amino acid sequences, which may reduce the risk of rejection by the human immune system as compared to, e.g., TCRs, polypeptides and proteins comprising mouse CDR and variable region amino acid sequences.

[0058] The phrase '‘antigenic specificity,” as used herein, means that the TCR can specifically bind to and immunologically recognize G12D RAS, G12V RAS, G13D RAS or Q61R RAS with high avidity. For example, a TCR may be considered to have “antigenic specificity” for G12D RAS, G12V RAS, G13D RAS or Q61R RAS if 1 x 104to 1 x 105T cells expressing the TCR secrete at least 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 foregoing values) of IFN-y upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with a low concentration of G12D RAS, G12V RAS, G13D RAS or Q61R RASpeptide (e.g.. 0.05 ng / mL to 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 foregoing values) or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding G12D RAS, G12V RAS, G13D RAS or Q61R RAS has been introduced such that the target cell expresses G12D RAS. G12V RAS, G13D RAS or Q61R RAS. respectively. Cells expressing the inventive TCRs may also secrete IFN-y upon co-culture with antigen-negative, applicable HLA molecule positive target cells pulsed with higher concentrations of G12D RAS, G12V RAS, G13D RAS or Q61R RAS peptide. The HLA molecule may be any of the HLA molecules described herein.

[0059] Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for G12D RAS, G12V RAS, G13D RAS or Q61R RAS if T cells expressing the TCR secrete at least twice (e.g., five times) as much IFN-y upon co-culture with (a) antigennegative. applicable HLA molecule positive target cells pulsed with a low concentration of G12D RAS, G12V RAS, G13D RAS or Q61R RAS peptide, respectively, or (b) antigennegative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding G12D RAS, G12V RAS, G13D RAS or Q61R RAS has been introduced such that the target cell expresses G12D RAS, G12V RAS, G13D RAS or Q61R RAS. respectively, as compared to the amount of IFN-y expressed by a negative control. The negative control may be, for example, (i) T cells expressing the TCR, co-cultured with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide with a different sequence from the G12D RAS. G12V RAS, G13D RAS or Q61R RAS peptide) or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced such that the target cell expresses the irrelevant peptide, or (ii) untransduced T cells (e.g., derived from PBMC. which do not express the TCR) co-cultured with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with the same concentration of G12D RAS, G12V RAS, G13D RAS or Q61R RAS peptide or (b) antigennegative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding G12D RAS, G12V RAS, G13D RAS or Q61R RAS has been introduced such that the target cell expresses G12D RAS, G12V RAS, G13D RAS or Q61R RAS. respectively. The HLA molecule expressed by the target cells of the negative control would be the same HLA molecule expressed by the target cells that are co-cultured with the T cells being tested. The HLA molecule may be any of the HLA molecules described herein. IFN-y secretion maybe measured by methods known in the art such as, for example, enzyme-linked immunosorbent assay (ELISA).

[0060] Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for G12D RAS, G12V RAS, G13D RAS or Q61R RAS if at least twice (e.g., five times) as many of the numbers of T cells expressing the TCR secrete IFN-y upon co-culture with (a) antigen-negative, applicable HLA molecule positive target cells pulsed with a low concentration of G12D RAS, G12V RAS, G13D RAS or Q61R RAS peptide, respectively, or (b) antigen-negative, applicable HLA molecule positive target cells into which a nucleotide sequence encoding G12D RAS, G12V RAS, G13D RAS or Q61R RAS, respectively, has been introduced such that the target cell expresses G12D RAS, G12V RAS, G13D RAS or Q61R RAS, respectively, as compared to the numbers of negative control T cells that secrete IFN-y. The HLA molecule, concentration of peptide, and the negative control may be as described herein with respect to other aspects of the invention. The numbers of cells secreting IFN-y may be measured by methods known in the art such as, for example, ELISPOT.

[0061] Alternatively or additionally, a TCR may be considered to have “antigenic specificity” for G12D RAS, G12V RAS, G13D RAS or Q61R RAS if T cells expressing the TCR upregulate expression of one or more T-cell activation markers as measured by, for example, flow cytometry after stimulation with target cells expressing G12D RAS, G12V RAS, G13D RAS or Q61R RAS, respectively. Examples of T-cell activation markers include 4-1BB, 0X40, CD107a, CD69, and cytokines that are upregulated upon antigen stimulation (e.g., tumor necrosis factor (TNF). interleukin (IL)-2, etc.).

[0062] An aspect of the invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as an alpha (a) chain of a TCR, a beta ( ) chain of a TCR, a gamma (y) chain of a TCR, a delta (6) chain of a TCR, or a combination thereof. The polypeptides of the inventive TCR can comprise any amino acid sequence, provided that the TCR has antigenic specificity for G12D RAS, G12V RAS, G13D RAS or Q61R RAS. In some aspects, the TCR is non-naturally occurring.

[0063] In an aspect of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising a complementarity determining region (CDR)l, a CDR2, and a CDR3 of a TCR. In an aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 1 (CDR1 of a chain of 4342 TCR 8), a CDR2 comprising the amino acid sequence ofSEQ ID NO: 2 (CDR2 of a chain of 4342 TCR 8), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 3 (CDR3 of a chain of 4342 TCR 8), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 4 (CDR1 of P chain of 4342 TCR 8), a CDR2 comprising the amino acid sequence of SEQ ID NO: 5 (CDR2 of chain of 4342 TCR 8), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 6 (CDR3 of P chain of 4342 TCR 8).

[0064] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 11 (CDR1 of a chain of 4400 TCR 5), a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 (CDR2 of a chain of 4400 TCR 5), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13 (CDR3 of a chain of 4400 TCR 5), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 14 (CDR1 of P chain of 4400 TCR 5), a CDR2 comprising the amino acid sequence of SEQ ID NO: 15 (CDR2 of P chain of 4400 TCR 5), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 16 (CDR3 of p chain of 4400 TCR 5).

[0065] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 21 (CDR1 of a chain of 4400 TCR 11), a CDR2 comprising the amino acid sequence of SEQ ID NO: 22 (CDR2 of a chain of 4400 TCR 11). and a CDR3 comprising the amino acid sequence of SEQ ID NO: 23 (CDR3 of a chain of 4400 TCR 11), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 24 (CDR1 of P chain of 4400 TCR 11), a CDR2 comprising the amino acid sequence of SEQ ID NO: 25 (CDR2 of chain of 4400 TCR 11 ). and a CDR3 comprising the amino acid sequence of SEQ ID NO: 26 (CDR3 of p chain of 4400 TCR 11 ).

[0066] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 31 (CDR1 of a chain of 4400 TCR 37), a CDR2 comprising the amino acid sequence of SEQ ID NO: 32 (CDR2 of a chain of 4400 TCR 37), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 33 (CDR3 of a chain of 4400 TCR 37), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 34 (CDR1 of P chain of 4400 TCR 37), a CDR2 comprising the amino acid sequence of SEQ ID NO: 35 (CDR2 of chain of 4400 TCR 37). and a CDR3 comprising the amino acid sequence of SEQ ID NO: 36 (CDR3 of p chain of 4400 TCR 37).

[0067] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 41 (CDR1 of a chain of 4400 TCR 47), a CDR2 comprising the amino acid sequence of SEQ ID NO: 42 (CDR2 of a chain of 4400 TCR 47), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 43 (CDR3 of a chain of 4400 TCR 47), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 44 (CDR1 of (3 chain of 4400 TCR 47), a CDR2 comprising the amino acid sequence of SEQ ID NO: 45 (CDR2 of ( chain of 4400 TCR 47), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 46 (CDR3 of chain of 4400 TCR 47).

[0068] In an aspect of the invention, the TCR comprises two polypeptide chains, each of which comprises a variable region comprising a CDR1, a CDR2, and a CDR3 of a TCR. In an aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 101 (CDR1 of a chain of 4589 NEO4 TCR 1), a CDR2 comprising the amino acid sequence of SEQ ID NO: 102 (CDR2 of a chain of 4589 NEO4 TCR 1), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 103 (CDR3 of a chain of 4589 NEO4 TCR 1), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 104 (CDR1 of P chain of 4589 NEO4 TCR 1). a CDR2 comprising the amino acid sequence of SEQ ID NO: 105 (CDR2 of P chain of 4589 NEO4 TCR 1), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 106 (CDR3 of p chain of 4589 NEO4 TCR 1).

[0069] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 111 (CDR1 of a chain of 4424 TCR 11), a CDR2 comprising the amino acid sequence of SEQ ID NO: 112 (CDR2 of a chain of 4424 TCR 1 1), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 113 (CDR3 of a chain of 4424 TCR 11), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 114 (CDR1 of P chain of 4424 TCR 11), a CDR2 comprising the amino acid sequence of SEQ ID NO: 115 (CDR2 of P chain of 4424 TCR 11), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 116 (CDR3 of p chain of 4424 TCR 11).

[0070] In another aspect of the invention, the TCR comprises a first poly peptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 121 (CDR1 of a chain of 4424 TCR 12), a CDR2 comprising the amino acid sequence of SEQ ID NO: 122 (CDR2 of a chain of 4424 TCR 12), and a CDR3 comprising the amino acid sequence ofSEQ ID NO: 123 (CDR3 of a chain of 4424 TCR 12), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 124 (CDR1 of p chain of 4424 TCR 12), a CDR2 comprising the amino acid sequence of SEQ ID NO: 125 (CDR2 of P chain of 4424 TCR 12), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 126 (CDR3 of p chain of 4424 TCR 12).

[0071] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 131 (CDR1 of a chain of 4424 TCR 13B), a CDR2 comprising the amino acid sequence of SEQ ID NO: 132 (CDR2 of a chain of 4424 TCR 13B), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 133 (CDR3 of a chain of 4424 TCR 13B). and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 134 (CDR1 of P chain of 4424 TCR 13B), a CDR2 comprising the amino acid sequence of SEQ ID NO: 135 (CDR2 of P chain of 4424 TCR 13B), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 136 (CDR3 of p chain of 4424 TCR 13B).

[0072] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 141 (CDR1 of a chain of 4424 TCR 17), a CDR2 comprising the amino acid sequence of SEQ ID NO: 142 (CDR2 of a chain of 4424 TCR 17), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 143 (CDR3 of a chain of 4424 TCR 17), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 144 (CDR1 of P chain of 4424 TCR 17), a CDR2 comprising the amino acid sequence of SEQ ID NO: 145 (CDR2 of P chain of 4424 TCR 17), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 146 (CDR3 of p chain of 4424 TCR 17).

[0073] In another aspect of the invention, the TCR comprises a first polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 151 (CDR1 of a chain of 4424 TCR 18), a CDR2 comprising the amino acid sequence of SEQ ID NO: 152 (CDR2 of a chain of 4424 TCR 18). and a CDR3 comprising the amino acid sequence of SEQ ID NO: 153 (CDR3 of a chain of 4424 TCR 18), and a second polypeptide chain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 154 (CDR1 of P chain of 4424 TCR 18), a CDR2 comprising the amino acid sequence of SEQ ID NO: 155 (CDR2 of P chain of 4424 TCR 18), and a CDR3 comprising the amino acid sequence of SEQ ID NO: 156 (CDR3 of P chain of 4424 TCR 18).

[0074] In this regard, the inventive TCR can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 -6, 1 1 -16, 21-26, 31 -36, 41- 46, 101-106, 111-116, 121-126, 131-136, 141-146, and 151-156. In an aspect of the invention, the TCR comprises the amino acid sequences of: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 1-6, (d) all of SEQ ID NOs: 11-13, (e) all of SEQ ID NOs: 14-16, (f) all of SEQ ID NOs: 11-16, (g) all of SEQ ID NOs: 21-23, (h) all of SEQ ID NOs: 24-26, (i) all of SEQ ID NOs: 21-26, (j) all of SEQ ID NOs: 31-33, (k) all of SEQ ID NOs: 34-36, (1) all of SEQ ID NOs: 31-36, (m) all of SEQ ID NOs: 41-43, (n) all of SEQ ID NOs: 44-46. (o) all of SEQ ID NOs: 41-46. (p) all of SEQ ID NOs: 101-103, (q) all of SEQ ID NOs: 104-106. (r) all of SEQ ID NOs: 101-106. (s) all of SEQ ID NOs: 1 11-113. (t) all of SEQ ID NOs: 1 14-116, (u) all of SEQ ID NOs: 111-116, (v) all of SEQ ID NOs: 121-123, (w) all of SEQ ID NOs: 124-126, (x) all of SEQ ID NOs: 121-126, (y) all of SEQ ID NOs: 131-133, (z) all of SEQ ID NOs: 134-136, (aa) all of SEQ ID NOs: 131-136, (bb) all of SEQ ID NOs: 141-143, (cc) all of SEQ ID NOs: 144-146, (dd) all of SEQ ID NOs: 141- 146, (ee) all of SEQ ID NOs: 151-153, (ff) all of SEQ ID NOs: 154-156, or (gg) all of SEQ ID NOs: 151-156. In an especially preferred aspect, the TCR comprises the amino acid sequences of: (a) all of SEQ ID NOs: 1-6, (b) all of SEQ ID NOs: 11-16, (c) all of SEQ ID NOs: 21-26, (d) all of SEQ ID NOs: 31-36, (e) all of SEQ ID NOs: 41-46, (f) all of SEQ ID NOs: 101-106. (g) all of SEQ ID NOs: 111-116, (h) all of SEQ ID NOs: 121-126, (i) all of SEQ ID NOs: 131-136, (j) all of SEQ ID NOs: 141-146, or (k) all of SEQ ID NOs: 151-156.

[0075] In an aspect of the invention, the TCR comprises an amino acid sequence of a variable region of a TCR comprising the CDRs set forth above. In this regard, the TCR can comprise the amino acid sequence of: (1) SEQ ID NO: 7 (predicted sequence of variable region of a chain of 4342 TCR 8 without N-terminal signal peptide); (2) SEQ ID NO: 8 (predicted sequence of variable region of 0 chain of 4342 TCR 8 without N-terminal signal peptide); (3) SEQ ID NO: 9 (variable region of a chain of 4342 TCR 8 with N-terminal signal peptide); (4) SEQ ID NO: 10 (variable region of (3 chain of 4342 TCR 8 with N-terminal signal peptide); (5) SEQ ID NO: 17 (predicted sequence of variable region of a chain of 4400 TCR 5 without N-terminal signal peptide); (6) SEQ ID NO: 18 (predicted sequence of variable region of 0 chain of 4400 TCR 5 without N-terminal signal peptide); (7) SEQ ID NO: 19 (variable region of a chain of 4400 TCR 5 with N-terminal signal peptide); (8) SEQ ID NO: 20 (variable region of 0 chain of 4400 TCR 5 with N-terminal signal peptide); (9) SEQ ID NO: 27 (predicted sequence of variable region of a chain of 4400 TCR 11 withoutN-terminal signal peptide); (10) SEQ ID NO: 28 (predicted sequence of variable region of P chain of 4400 TCR 11 without N-terminal signal peptide); (11) SEQ ID NO: 29 (variable region of a chain of 4400 TCR 11 with N-terminal signal peptide); (12) SEQ ID NO: 30 (variable region of chain of 4400 TCR 11 with N-terminal signal peptide); (13) SEQ ID NO: 37 (predicted sequence of variable region of a chain of 4400 TCR 37 without N-terminal signal peptide); (14) SEQ ID NO: 38 (predicted sequence of variable region of P chain of 4400 TCR 37 without N-terminal signal peptide); (15) SEQ ID NO: 39 (variable region of a chain of 4400 TCR 37 with N-terminal signal peptide); (16) SEQ ID NO: 40 (variable region of P chain of 4400 TCR 37 with N-terminal signal peptide); (17) SEQ ID NO: 47 (predicted sequence of variable region of a chain of 4400 TCR 47 without N-terminal signal peptide); (18) SEQ ID NO: 48 (predicted sequence of variable region of P chain of 4400 TCR 47 without N-terminal signal peptide); (19) SEQ ID NO: 49 (variable region of a chain of 4400 TCR 47 with N-terminal signal peptide); (20) SEQ ID NO: 50 (variable region of P chain of 4400 TCR 47 with N-terminal signal peptide); (21) SEQ ID NO: 107 (predicted sequence of variable region of a chain of 4589 NEO4 TCR 1 without N-terminal signal peptide); (22) SEQ ID NO: 108 (predicted sequence of variable region of P chain of 4589 NEO4 TCR 1 without N-terminal signal peptide); (23) SEQ ID NO: 109 (variable region of a chain of 4589 NEO4 TCR 1 with N-terminal signal peptide); (24) SEQ ID NO: 110 (variable region of P chain of 4589 NEO4 TCR 1 with N-terminal signal peptide); (25) SEQ ID NO: 117 (predicted sequence of variable region of a chain of 4424 TCR 11 without N-terminal signal peptide); (26) SEQ ID NO: 118 (predicted sequence of variable region of P chain of 4424 TCR 11 without N-terminal signal peptide); (27) SEQ ID NO: 119 (variable region of a chain of 4424 TCR 11 with N-terminal signal peptide); (28) SEQ ID NO: 120 (variable region of P chain of 4424 TCR 11 with N-terminal signal peptide); (29) SEQ ID NO: 127 (predicted sequence of variable region of a chain of 4424 TCR 12 without N-terminal signal peptide); (30) SEQ ID NO: 128 (predicted sequence of variable region of P chain of 4424 TCR 12 without N-terminal signal peptide); (31) SEQ ID NO: 129 (variable region of a chain of 4424 TCR 12 with N-terminal signal peptide); (32) SEQ ID NO: 130 (variable region of P chain of 4424 TCR 12 with N-terminal signal peptide); (33) SEQ ID NO: 137 (predicted sequence of variable region of a chain of 4424 TCR 13B without N-terminal signal peptide); (34) SEQ ID NO: 138 (predicted sequence of variable region of P chain of 4424 TCR 13B without N- terminal signal peptide); (35) SEQ ID NO: 139 (variable region of a chain of 4424 TCR 13B with N-terminal signal peptide); (36) SEQ ID NO: 140 (variable region of P chain of 4424TCR 13B with N-terminal signal peptide): (37) SEQ ID NO: 147 (predicted sequence of variable region of a chain of 4424 TCR 17 without N-terminal signal peptide); (38) SEQ ID NO: 148 (predicted sequence of variable region of 0 chain of 4424 TCR 17 without N- terminal signal peptide); (39) SEQ ID NO: 149 (variable region of a chain of 4424 TCR 17 with N-terminal signal peptide); (40) SEQ ID NO: 150 (variable region of 0 chain of 4424 TCR 17 with N-terminal signal peptide); (41) SEQ ID NO: 157 (predicted sequence of variable region of a chain of 4424 TCR 18 without N-terminal signal peptide); (42) SEQ ID NO: 158 (predicted sequence of variable region of 0 chain of 4424 TCR 18 without N- terminal signal peptide); (43) SEQ ID NO: 159 (variable region of a chain of 4424 TCR 18 with N-terminal signal peptide); (44) SEQ ID NO: 160 (variable region of 0 chain of 4424 TCR 18 with N-terminal signal peptide); (45) both of SEQ ID NOs: 7 and 8; (46) both of SEQ ID NOs: 9 and 10; (47) both of SEQ ID NOs: 17 and 18; (48) both of SEQ ID NOs: 19 and 20: (49) both of SEQ ID NOs: 27 and 28; (50) both of SEQ ID NOs: 29 and 30; (51) both of SEQ ID NOs: 37 and 38; (52) both of SEQ ID NOs: 39 and 40; (53) both of SEQ ID NOs: 47 and 48, (54) both of SEQ ID NOs: 49 and 50; (55) both of SEQ ID NOs: 107 and 108; (56) both of SEQ ID NOs: 109 and 110; (57) both of SEQ ID NOs: 117 and 118; (58) both of SEQ ID NOs: 119 and 120; (59) both of SEQ ID NOs: 127 and 128; (60) both of SEQ ID NOs: 129 and 130; (61) both of SEQ ID NOs: 137 and 138; (62) both of SEQ ID NOs: 139 and 140; (63) both of SEQ ID NOs: 147 and 148, (64) both of SEQ ID NOs: 149 and 150; (65) both of SEQ ID NOs: 157 and 158; or (66) both of SEQ ID NOs: 159 and 160.

[0076] Preferably, the TCR comprises the amino acid sequences of (i) both of SEQ ID NOs: 7 and 8, (ii) both of SEQ ID NOs: 9 and 10, (iii) both of SEQ ID NOs: 17 and 18, (iv) both of SEQ ID NOs: 19 and 20. (v) both of SEQ ID NOs: 27 and 28, (vi) both of SEQ ID NOs: 29 and 30, (vii) both of SEQ ID NOs: 37 and 38, (viii) both of SEQ ID NOs: 39 and 40, (ix) both of SEQ ID NOs: 47 and 48, (x) both of SEQ ID NOs: 49 and 50, (xi) both of SEQ ID NOs: 107 and 108; (xii) both of SEQ ID NOs: 109 and 110; (xiii) both of SEQ ID NOs: 117 and 118; (xiv) both of SEQ ID NOs: 119 and 120; (xv) both of SEQ ID NOs: 127 and 128; (xvi) both of SEQ ID NOs: 129 and 130; (xvii) both of SEQ ID NOs: 137 and 138; (xviii) both of SEQ ID NOs: 139 and 140; (xix) both of SEQ ID NOs: 147 and 148, (xx) both of SEQ ID NOs: 149 and 150; (xxi) both of SEQ ID NOs: 157 and 158; or (xxii) both of SEQ ID NOs: 159 and 160.

[0077] The inventive TCRs may further comprise an a chain constant region and a 0 chain constant region. The constant region may be derived from any suitable species such as,e.g., human or mouse. In an aspect of the invention, the TCRs further comprise murine a and P chain constant regions or human a and chain constant regions. As used herein, the term “murine” or “human,” when referring to a TCR or any component of a TCR described herein (e.g., CDR, variable region, constant region, a chain, and / or P chain), means a TCR (or component thereof) which is derived from a mouse or a human, respectively, i.e., a TCR (or component thereof) that originated from or was, at one time, expressed by a mouse T cell or a human T cell, respectively.

[0078] An aspect of the invention provides a chimeric TCR comprising a human variable region and a murine constant region, wherein the TCR has antigenic specificity for a mutated human RAS amino acid sequence with a substitution of (i) glycine at position 12 with aspartic acid, (ii) glycine at position 12 with valine, (iiii) glycine at position 13 with aspartic acid or (iv) glutamine at position 61 with arginine. The murine constant region may provide any one or more advantages. For example, the murine constant region may diminish mispairing of the inventive TCR with the endogenous TCRs of the host cell into which the inventive TCR is introduced. Alternatively or additionally, the murine constant region may increase expression of the inventive TCR as compared to the same TCR with a human constant region. The chimeric TCR may comprise the amino acid sequence of SEQ ID NO: 67 (WT murine a chain constant region), SEQ ID NO: 68 (WT murine P chain constant region), or both SEQ ID NOs: 67 and 68. Preferably, the inventive TCR comprises the amino acid sequences of both of SEQ ID NOs: 67 and 68. The chimeric TCR may comprise any of the murine constant regions described herein in combination with any of the CDR regions as described herein with respect to other aspects of the invention. In this regard, the TCR may comprise the amino acid sequences of: (a) all of SEQ ID NOs: 1-3 and 67. (b) all of SEQ ID NOs: 4-6 and 68, (c) all of SEQ ID NOs: 1-6 and 67-68, (d) all of SEQ ID NOs: 1 1-13 and67, (e) all of SEQ ID NOs: 14-16 and 68, (f) all of SEQ ID NOs: 11-16 and 67-68, (g) all of SEQ ID NOs: 21-23 and 67, (h) all of SEQ ID NOs: 24-26 and 68, (i) all of SEQ ID NOs: 21- 26 and 67-68, (j) all of SEQ ID NOs: 31-33 and 67, (k) all of SEQ ID NOs: 34-36 and 68, (1) all of SEQ ID NOs: 31-36 and 67-68, (m) all of SEQ ID NOs: 41-43 and 67, (n) all of SEQ ID NOs: 44-46 and 68, (o) all of SEQ ID NOs: 41-46 and 67-68, (p) all of SEQ ID NOs: 101- 103 and 67, (q) all of SEQ ID NOs: 104-106 and 68, (r) all of SEQ ID NOs: 101-106 and 67-68, (s) all of SEQ ID NOs: 111-113 and 67, (t) all of SEQ ID NOs: 114-116 and 68, (u) all of SEQ ID NOs: 111-116 and 67-68. (v) all of SEQ ID NOs: 121-123 and 67. (w) all of SEQ ID NOs: 124-126 and 68, (x) all of SEQ ID NOs: 121-126 and 67-68, (y) all of SEQ ID NOs:131-133 and 67, (z) all of SEQ ID NOs: 134-136 and 68, (aa) all of SEQ ID NOs: 131-136 and 67-68, (bb) all of SEQ ID NOs: 141 -143 and 67, (cc) all of SEQ ID NOs: 144-146 and 68, (dd) all of SEQ ID NOs: 141-146 and 67-68, (ee) all of SEQ ID NOs: 151-153 and 67, (ff) all of SEQ ID NOs: 154-156 and 68, or (gg) all of SEQ ID NOs: 151-156 and 67-68.

[0079] In another aspect 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 the amino acid sequences of: (1) both of SEQ ID NOs: 7 and 67, (2) both of SEQ ID NOs: 8 and 68, (3) both of SEQ ID NOs: 9 and 67, (4) both of SEQ ID NOs: 10 and 68, (5) both of SEQ ID NOs: 17 and 67. (6) both of SEQ ID NOs: 18 and 68. (7) both of SEQ ID NOs: 19 and 67, (8) both of SEQ ID NOs: 20 and 68, (9) both of SEQ ID NOs: 27 and 67, (10) both of SEQ ID NOs: 28 and 68, (11) both of SEQ ID NOs: 29 and 67, (12) both of SEQ ID NOs: 30 and 68, (13) both of SEQ ID NOs: 37 and 67. (14) both of SEQ ID NOs: 38 and 68, (15) both of SEQ ID NOs: 39 and 67, (16) both of SEQ ID NOs: 40 and 68, (17) both of SEQ ID NOs: 47 and 67, (17) both of SEQ ID NOs: 48 and 68, (19) both of SEQ ID NOs: 49 and 67, (20) both of SEQ ID NOs: 50 and 68, (21) both of SEQ ID NOs: 107 and 67, (22) both of SEQ ID NOs: 108 and 68, (23) both of SEQ ID NOs: 109 and 67. (24) both of SEQ ID NOs: 110 and 68. (25) both of SEQ ID NOs: 117 and 67, (26) both of SEQ ID NOs: 118 and 68. (27) both of SEQ ID NOs: 119 and 67, (28) both of SEQ ID NOs: 120 and 68. (29) both of SEQ ID NOs: 127 and 67, (30) both of SEQ ID NOs: 128 and 68, (31) both of SEQ ID NOs: 129 and 67, (32) both of SEQ ID NOs: 130 and 68, (33) both of SEQ ID NOs: 137 and 67. (34) both of SEQ ID NOs: 138 and 68, (35) both of SEQ ID NOs: 139 and 67, (36) both of SEQ ID NOs: 140 and 68, (37) both of SEQ ID NOs: 147 and 67, (38) both of SEQ ID NOs: 148 and 68, (39) both of SEQ ID NOs: 149 and 67, (40) both of SEQ ID NOs: 150 and 68, (41) both of SEQ ID NOs: 157 and 67, (42) both of SEQ ID NOs: 158 and 68, (43) both of SEQ ID NOs: 159 and 67, (44) both of SEQ ID NOs: 160 and 68, (45) all of SEQ ID NOs: 7-8 and 67-68. (46) all of SEQ ID NOs: 9-10 and 67-68, (47) all of SEQ ID NOs: 17- 18 and 67-68, (48) all of SEQ ID NOs: 19-20 and 67-68, (49) all of SEQ ID NOs: 27-28 and 67-68, (50) all of SEQ ID NOs: 29-30 and 67-68, (51) all of SEQ ID NOs: 37-38 and 67-68, (52) all of SEQ ID NOs: 39-40 and 67-68, (53) all of SEQ ID NOs: 47-48 and 67-68, (54) all of SEQ ID NOs: 49-50 and 67-68, (55) all of SEQ ID NOs: 107-108 and 67-68, (56) all of SEQ ID NOs: 109-110 and 67-68. (57) all of SEQ ID NOs: 117-118 and 67-68, (58) all of SEQ ID NOs: 119-120 and 67-68, (59) all of SEQ ID NOs: 127-128 and 67-68, (60) all ofSEQ ID NOs: 129-130 and 67-68. (61) all of SEQ ID NOs: 137-138 and 67-68, (62) all ofSEQ ID NOs: 139-140 and 67-68, (63) all of SEQ ID NOs: 147-148 and 67-68, (64) all ofSEQ ID NOs: 149-150 and 67-68, (65) all of SEQ ID NO: 157-158 and 67-38, or (66) all ofSEQ ID NOs: 159-160 and 67-68.

[0080] In an aspect 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 with one, two, three, or four amino acid substitution(s) in the constant region of one or both of the a and P chain. 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 a and chains. In an especially preferred aspect, the TCR comprises a murine constant region with one, two, three, or four amino acid substitution(s) in the murine constant region of the a chain and one amino acid substitution in the murine constant region of the P chain. In some aspects, the TCRs comprising the substituted constant region advantageously provide one or more of increased recognition of G12D RAS+, G12V RAS+. G13D RAS+or Q61R RAS+targets, increased expression by a host cell, diminished mispairing with endogenous TCRs, and increased anti-tumor activity as compared to the parent TCR comprising an unsubstituted (wild-ty pe) constant region. In general, the substituted amino acid sequences of the murine constant regions of the TCR a and P chains, SEQ ID NOs: 63 and 64. respectively, correspond with all or portions of the unsubstituted murine constant region amino acid sequences SEQ ID NOs: 67 and 68, respectively, with SEQ ID NO: 63 having one, two, three, or four amino acid substitution(s) when compared to SEQ ID NO: 67 and SEQ ID NO: 64 having one amino acid substitution when compared to SEQ ID NO: 68. In this regard, an aspect of the invention provides a TCR comprising the amino acid sequences of (a) SEQ ID NO: 63 (constant region of a chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Ala, Vai, Leu, He, Pro, Phe, Met, or Trp; (iii) X at position 114 is Met, Ala. Vai, Leu, He, Pro, Phe, or Trp; and (iv) X at position 115 is Gly, Ala, Vai, Leu, He, Pro, Phe. Met, or Trp; (b) SEQ ID NO: 64 (constant region of P chain), wherein X at position 57 is Ser or Cys; or (c) both of SEQ ID NOs: 63 and 64. In an aspect of the invention, the TCR comprising SEQ ID NO: 63 does not comprise SEQ ID NO: 67 (unsubstituted murine constant region of a chain). In an aspect of the invention, the TCR comprising SEQ ID NO: 64 does not comprise SEQ ID NO: 68 (unsubstituted murine constant region of P chain).

[0081] In an aspect of the invention, the substituted constant region includes cysteine substitutions in the constant region of one or both of the a and P chains to provide a cysteine-substituted TCR. Opposing cysteines in the a and the P chains provide a disulfide bond that links the constant regions of the a and the chains of the substituted TCR to one another and which is not present in a TCR comprising the unsubstituted murine 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 (Thr48) of SEQ ID NO: 67 and the native Ser at position 57 (Ser57) of SEQ ID NO: 68 may be substituted with Cys. Preferably, both of the native Thr48 of SEQ ID NO: 67 and the native Ser57 of SEQ ID NO: 68 are substituted with Cys. Examples of cysteinesubstituted TCR constant regions sequences are set forth in Table 3. In an aspect of the invention, the cysteine-substituted TCR comprises (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) both of SEQ ID NOs: 63 and 64. wherein both of SEQ ID NOs: 63 and 64 are as defined in Table 3. The cysteine-substituted TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.TABLE 3

[0082] In an aspect of the invention, the substituted amino acid sequence includes substitutions of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of the a chain with a hydrophobic amino acid 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 may increase the hydrophobicity of the TM domain of the TCR as compared to a TCR that lacks the 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 the native Seri 12, Metl 14, and Gly 115 of SEQ ID NO: 67 may, independently, be substituted with Ala. Vai, Leu. He. Pro, Phe. Met. or Trp; preferably with Leu, He, or Vai. Preferably, all three of the native Seri 12, Metl 14, and Glyl 15 of SEQ ID NO: 67 may, independently, be substituted with Ala, Vai, Leu, He, Pro,Phe, Met, or Trp; preferably with Leu. He. or Vai. In an aspect of the invention, the LVL- modified TCR comprises (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) both of SEQ ID NOs: 63 and 64, wherein both of SEQ ID NOs: 63 and 64 are as defined in Table 4. The LVL-modified TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.TABLE 4

[0083] In an aspect of the invention, the substituted amino acid sequence includes the cysteine substitutions in the constant region of one or both of the a and (3 chains in combination with the substitution(s) of one, two, or three amino acids in the transmembrane (TM) domain of the constant region of the a chain with a hydrophobic amino acid (also referred to herein as “cysteine-substituted. LVL-modified TCR ”). In this regard, the TCR is a cysteine-substituted, LVL-modified, chimeric TCR in which the native Thr48 of SEQ ID NO: 67 is substituted with Cys; one, two, or three of the native Seri 12, Metl 14, and Gly 115 of SEQ ID NO: 67 are. independently, substituted with Ala. Vai, Leu, He, Pro, Phe, Met, or Trp; preferably with Leu, He, or Vai; and the native Ser57 of SEQ ID NO: 68 is substituted with Cys. Preferably, all three of the native Seri 12, Metl 14, and Gly 115 of SEQ ID NO: 67 may, independently, be substituted with Ala, Vai, Leu, He, Pro, Phe, Met, or Trp; preferably with Leu, He, or Vai. In an aspect of the invention, the cysteine-substituted, LVL-modifiedTCR comprises (i) SEQ ID NO: 63, (ii) SEQ ID NO: 64, or (iii) both of SEQ ID NOs: 63 and 64, wherein both of SEQ ID NOs: 63 and 64 are as defined in Table 5. The cysteinesubstituted, LVL-modified TCRs of the invention may include the substituted constant region in addition to any of the CDRs or variable regions described herein.TABLE 5

[0084] In an aspect of the invention, the cysteine-substituted, LVL-modified TCR comprises (a) SEQ ID NO: 65 (a chain constant region of cysteine-substituted. LVL- modified TCR); (b) SEQ ID NO: 66 (|3 chain constant region of cysteine-substituted, LVL- modified TCR); or (c) both (a) and (b).

[0085] In an aspect, the cysteine-substituted, LVL-modified TCR comprises a full-length a chain and a full-length [3 chain. Examples of cysteine-substituted, LVL-modified TCR a chain and [3 chain sequences are set forth in Table 10 and Table 30. In this regard, in an aspect of the invention, the TCR comprises the amino acid sequence(s) of (1) SEQ ID NO: 69, (2) SEQ ID NO: 70, (3) SEQ ID NO: 71, (4) SEQ ID NO: 72, (5) SEQ ID NO: 73, (6) SEQ ID NO: 74, (7) SEQ ID NO: 75, (8) SEQ ID NO: 76, (9) SEQ ID NO: 77. (10) SEQ ID NO: 78, (11) SEQ ID NO: 79, (12) SEQ ID NO: 80, (13) SEQ ID NO: 81, (14) SEQ ID NO: 82, (15) SEQ ID NO: 83, (16) SEQ ID NO: 84, (17) SEQ ID NO: 85, (18) SEQ ID NO: 86, (19) SEQ ID NO: 87, (20) SEQ ID NO: 88, (21) SEQ ID NO: 161, (22) SEQ ID NO: 162,(23) SEQ ID NO: 163. (24) SEQ ID NO: 164. (25) SEQ ID NO: 165. (26) SEQ ID NO: 166.(27) SEQ ID NO: 167, (28) SEQ ID NO: 168, (29) SEQ ID NO: 169, (30) SEQ ID NO: 170,(31) SEQ ID NO: 171, (32) SEQ ID NO: 172, (33) SEQ ID NO: 173, (34) SEQ ID NO: 174,(35) SEQ ID NO: 175, (36) SEQ ID NO: 176, (37) SEQ ID NO: 177, (38) SEQ ID NO: 178,(39) SEQ ID NO: 179, (40) SEQ ID NO: 180. (41) SEQ ID NO: 181. (42) SEQ ID NO: 182.(43) SEQ ID NO: 183, (44) SEQ ID NO: 184, (45) both of SEQ ID NOs: 69 and 70, (46) both of SEQ ID NOs: 71 and 72, (47) both of SEQ ID NOs: 73 and 74, (48) both of SEQ ID NOs: 75 and 76, (49) both of SEQ ID NOs: 77 and 78, (50) both of SEQ ID NOs: 79 and 80, (51) both of SEQ ID NOs: 81 and 82, (52) both of SEQ ID NOs: 83 and 84, (53) both of SEQ ID NOs: 85 and 86, (54) both of SEQ ID NOs: 87 and 88. (55) both of SEQ ID NOs: 161 and 1 2, (56) both of SEQ ID NOs: 163 and 1 4, (57) both of SEQ ID NOs: 165 and 1 6, (58) both of SEQ ID NOs: 167 and 168, (59) both of SEQ ID NOs: 169 and 170, (60) both of SEQ ID NOs: 171 and 172, (61) both of SEQ ID NOs: 173 and 174, (62) both of SEQ ID NOs: 175 and 176. (63) both of SEQ ID NOs: 177 and 178. (64) both of SEQ ID NOs: 179 and 180, (65) both of SEQ ID NOs: 181 and 182, or (66) both of SEQ ID NOs: 183 and 184.

[0086] Also provided by the invention is a polypeptide 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 connected by one or more peptide bonds.

[0087] With respect to the inventive polypeptides, the functional portion can be any portion comprising contiguous amino acids of the TCR of which it is a part, provided that the functional portion specifically binds to G12D RAS, G12V RAS, G13D RAS or Q61R RAS. The term “functional portion,” when used in reference to a TCR, refers to any part or fragment of the TCR of the invention, which part or fragment retains the biological activity of the TCR of which it is a part (the parent TCR). Functional portions encompass, for example, those parts of a TCR that retain the ability to specifically bind to G12D RAS. G12V RAS, G13D RAS or Q61R RAS (e.g., within the context of any of the HLA molecules described herein), or detect, treat, or prevent cancer, to a similar extent, the same extent, or to a higher extent, as the parent TCR. In reference to the parent TCR, the functional portion can comprise, for instance, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more, of the parent TCR.

[0088] The functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are notfound 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., specifically binding to G12D RAS, G12V RAS, G13D RAS or Q61R RAS; and / or having the ability to detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent TCR.

[0089] The polypeptide can comprise a functional portion of either or both of the a and 0 chains of the TCRs of the invention, such as a functional portion comprising one or more of the CDR1, CDR2, and CDR3 of the variable region(s) of the a chain and / or 0 chain of a TCR of the invention. In an aspect of the invention, the polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (CDR1 of a chain of 4342 TCR 8), SEQ ID NO: 2 (CDR2 of a chain of 4342 TCR 8), SEQ ID NO: 3 (CDR3 of a chain of 4342 TCR 8), SEQ ID NO: 4 (CDR1 of 0 chain of 4342 TCR 8), SEQ ID NO: 5 (CDR2 of 0 chain of 4342 TCR 8), SEQ ID NO: 6 (CDR3 of 0 chain of 4342 TCR 8), SEQ ID NO: 11 (CDR1 of a chain of 4400 TCR 5), SEQ ID NO: 12 (CDR2 of a chain of 4400 TCR 5), SEQ ID NO: 13 (CDR3 of a chain of 4400 TCR 5), SEQ ID NO: 14 (CDR1 of 0 chain of 4400 TCR 5), SEQ ID NO: 15 (CDR2 of 0 chain of 4400 TCR 5), SEQ ID NO: 16 (CDR3 of 0 chain of 4400 TCR 5), SEQ ID NO: 21 (CDR1 of a chain of 4400 TCR 11), SEQ ID NO: 22 (CDR2 of a chain of 4400 TCR 11). SEQ ID NO: 23 (CDR3 of a chain of 4400 TCR 11), SEQ ID NO: 24 (CDR1 of 0 chain of 4400 TCR 11), SEQ ID NO: 25 (CDR2 of 0 chain of 4400 TCR 11), SEQ ID NO: 26 (CDR3 of 0 chain of 4400 TCR 11), SEQ ID NO: 31 (CDR1 of a chain of 4400 TCR 37), SEQ ID NO: 32 (CDR2 of a chain of 4400 TCR 37), SEQ ID NO: 33 (CDR3 of a chain of 4400 TCR 37), SEQ ID NO: 34 (CDR1 of 0 chain of 4400 TCR 37), SEQ ID NO: 35 (CDR2 of 0 chain of 4400 TCR 37). SEQ ID NO: 36 (CDR3 of 0 chain of 4400 TCR 37), SEQ ID NO: 41 (CDR1 of a chain of 4400 TCR 47), SEQ ID NO: 42 (CDR2 of a chain of 4400 TCR 47), SEQ ID NO: 43 (CDR3 of a chain of 4400 TCR 47), SEQ ID NO: 44 (CDR1 of 0 chain of 4400 TCR 47), SEQ ID NO: 45 (CDR2 of 0 chain of 4400 TCR 47), SEQ ID NO: 46 (CDR3 of 0 chain of 4400 TCR 47). SEQ ID NO: 101 (CDR1 of a chain of 4589 NEO4 TCR 1), SEQ ID NO: 102 (CDR2 of a chain of 4589 NEO4 TCR 1), SEQ ID NO: 103 (CDR3 of a chain of 4589 NEO4 TCR 1), SEQ ID NO: 104 (CDR1 of 0 chain of 4589 NEO4 TCR 1), SEQ ID NO: 105 (CDR2 of 0 chain of 4589 NEO4 TCR 1), SEQ ID NO: 106 (CDR3 of 0 chain of 4589 NEO4 TCR 1), SEQ ID NO: 111 (CDR1 of a chain of 4424 TCR 11), SEQ ID NO: 112 (CDR2 of a chain of 4424 TCR 11), SEQ ID NO: 113 (CDR3 of a chain of 4424 TCR 11), SEQ ID NO: 114 (CDR1 of 0 chain of 4424 TCR 11), SEQ ID NO: 115 (CDR2 ofP chain of 4424 TCR 11), SEQ ID NO: 116 (CDR3 of p chain of 4424 TCR 11), SEQ ID NO: 121 (CDR1 of a chain of 4424 TCR 12), SEQ ID NO: 122 (CDR2 of a chain of 4424 TCR 12), SEQ ID NO: 123 (CDR3 of a chain of 4424 TCR 12), SEQ ID NO: 124 (CDR1 of P chain of 4424 TCR 12), SEQ ID NO: 125 (CDR2 of P chain of 4424 TCR 12), SEQ ID NO: 126 (CDR3 of p chain of 4424 TCR 12), SEQ ID NO: 131 (CDR1 of a chain of 4424 TCR 13B), SEQ ID NO: 132 (CDR2 of a chain of 4424 TCR 13B), SEQ ID NO: 133 (CDR3 of a chain of 4424 TCR 13B), SEQ ID NO: 134 (CDR1 of P chain of 4424 TCR 13B), SEQ ID NO: 135 (CDR2 of P chain of 4424 TCR 13B), SEQ ID NO: 136 (CDR3 of P chain of 4424 TCR 13B), SEQ ID NO: 141 (CDR1 of a chain of 4424 TCR 17), SEQ ID NO: 142 (CDR2 of a chain of 4424 TCR 17). SEQ ID NO: 143 (CDR3 of a chain of 4424 TCR 17), SEQ ID NO: 144 (CDR1 of p chain of 4424 TCR 17), SEQ ID NO: 145 (CDR2 of P chain of 4424 TCR 17), SEQ ID NO: 146 (CDR3 of P chain of 4424 TCR 17), SEQ ID NO: 151 (CDR1 of a chain of 4424 TCR 18), SEQ ID NO: 152 (CDR2 of a chain of 4424 TCR 18), SEQ ID NO: 153 (CDR3 of a chain of 4424 TCR 18), SEQ ID NO: 154 (CDR1 of P chain of 4424 TCR 18), SEQ ID NO: 155 (CDR2 of P chain of 4424 TCR 18), SEQ ID NO: 156 (CDR3 of P chain of 4424 TCR 18), or a combination thereof. In this regard, the inventive polypeptide can comprise any one or more of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-6. 11-16, 21-26, 31-36. 41-46, 101-106, 111-116, 121- 126, 131-136, 141-146, and 151-156. In an aspect of the invention, the polypeptide comprises the amino acid sequences of: (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 1-6, (d) all of SEQ ID NOs: 11-13, (e) all of SEQ ID NOs: 14- 16, (f) all of SEQ ID NOs: 11-16, (g) all of SEQ ID NOs: 21-23, (h) all of SEQ ID NOs: 24- 26. (i) all of SEQ ID NOs: 21-26, (j) all of SEQ ID NOs: 31-33, (k) all of SEQ ID NOs: 34- 36, (1) all of SEQ ID NOs: 31-36, (m) all of SEQ ID NOs: 41 -43, (n) all of SEQ ID NOs: 44- 46, (o) all of SEQ ID NOs: 41-46, (p) all of SEQ ID NOs: 101-103, (q) all of SEQ ID NOs: 104-106, (r) all of SEQ ID NOs: 101-106, (s) all of SEQ ID NOs: 111-113, (t) all of SEQ ID NOs: 114-116, (u) all of SEQ ID NOs: 111-116, (v) all of SEQ ID NOs: 121-123, (w) all of SEQ ID NOs: 124-126, (x) all of SEQ ID NOs: 121-126, (y) all of SEQ ID NOs: 131-133, (z) all of SEQ ID NOs: 134-136, (aa) all of SEQ ID NOs: 131-136, (bb) all of SEQ ID NOs: 141-143, (cc) all of SEQ ID NOs: 144-146, (dd) all of SEQ ID NOs: 141-146, (ee) all of SEQ ID NO: 151-153. (ff) all of SEQ ID NO: 154-156. or (gg) all of SEQ ID NO: 151-156. In a preferred aspect, the polypeptide comprises the amino acid sequences of all of (i) SEQ ID NOs: 1-6, (li) SEQ ID NOs: 11-16, (lii) SEQ ID NOs: 21-26, (iv) SEQ ID NOs: 31-36, (v)SEQ ID NOs: 41-46. (vi) SEQ ID NOs: 101-106, (vii) SEQ ID NOs: 111-116, (viii) SEQ ID NOs: 121 -126, (ix) SEQ ID NOs: 131 -136, (x) SEQ ID NOs: 141 -146, or (xi) SEQ ID NO: 151-156.

[0090] In an aspect of the invention, the inventive polypeptide can comprise, for instance, the variable region of the inventive TCR comprising a combination of the CDR regions set forth above. In this regard, the polypeptide can comprise the amino acid sequence(s) of (1) SEQ ID NO: 7, (2) SEQ ID NO: 8, (3) SEQ ID NO: 9, (4) SEQ ID NO: 10, (5) SEQ ID NO: 17, (6) SEQ ID NO: 18, (7) SEQ ID NO: 19, (8) SEQ ID NO: 20, (9) SEQ ID NO: 27, (10) SEQ ID NO: 28, (11) SEQ ID NO: 29, (12) SEQ ID NO: 30, (13) SEQ ID NO: 37. (14) SEQ ID NO: 38. (15) SEQ ID NO: 39, (16) SEQ ID NO: 40. (17) SEQ ID NO: 47, (18) SEQ ID NO: 48, (19) SEQ ID NO: 49, (20) SEQ ID NO: 50, (21) SEQ ID NO: 107, (22) SEQ ID NO: 108, (23) SEQ ID NO: 109, (24) SEQ ID NO: 110, (25) SEQ ID NO: 117, (26) SEQ ID NO: 118, (27) SEQ ID NO: 119, (28) SEQ ID NO: 120, (29) SEQ ID NO: 127, (30) SEQ ID NO:128, (31) SEQ ID NO: 129, (32) SEQ ID NO: 130, (33) SEQ ID NO: 137, (34) SEQ ID NO:138, (35) SEQ ID NO: 139, (36) SEQ ID NO: 140, (37) SEQ ID NO: 147, (38) SEQ ID NO:148, (39) SEQ ID NO: 149, (40) SEQ ID NO: 150, (41) SEQ ID NO: 157, (42) SEQ ID NO:158, (43) SEQ ID NO: 159, (44) SEQ ID NO: 160, (45) both of SEQ ID NOs: 7 and 8, (46) both of SEQ ID NOs: 9 and 10, (47) both of SEQ ID NOs: 17 and 18, (48) both of SEQ ID NOs: 19 and 20, (49) both of SEQ ID NOs: 27 and 28, (50) both of SEQ ID NOs: 29 and 30. (51) both of SEQ ID NOs: 37 and 38, (52) both of SEQ ID NOs: 39 and 40, (53) both of SEQ ID NOs: 47 and 48, (54) both of SEQ ID NOs: 49 and 50, (55) both of SEQ ID NOs: 107 and 108, (56) both of SEQ ID NOs: 109 and 110, (57) both of SEQ ID NOs: 117 and 118, (58) both of SEQ ID NOs: 119 and 120, (59) both of SEQ ID NOs: 127 and 128, (60) both of SEQ ID NOs: 129 and 130, (61) both of SEQ ID NOs: 137 and 138, (62) both of SEQ ID NOs: 139 and 140, (63) both of SEQ ID NOs: 147 and 148, or (64) both of SEQ ID NOs: 149 and 150, (65) both of SEQ ID NO: 157 and 158, or (66) both of SEQ ID NO: 159 and 160. In a preferred aspect, the polypeptide comprises the amino acid sequences of (a) both of SEQ ID NOs: 7 and 8, (b) both of SEQ ID NOs: 9 and 10, (c) both of SEQ ID NOs: 17 and 18, (d) both of SEQ ID NOs: 19 and 20, (e) both of SEQ ID NOs: 27 and 28, (f) both of SEQ ID NOs: 29 and 30, (g) both of SEQ ID NOs: 37 and 38, (h) both of SEQ ID NOs: 39 and 40, (i) both of SEQ ID NOs: 47 and 48, (j) both of SEQ ID NOs: 49 and 50, (k) both of SEQ ID NOs: 107 and 108. (1) both of SEQ ID NOs: 109 and 110. (m) both of SEQ ID NOs: 117 and 1 18, (n) both of SEQ ID NOs: 119 and 120, (o) both of SEQ ID NOs: 127 and 128, (p) bothof SEQ ID NOs: 129 and 130. (q) both of SEQ ID NOs: 137 and 138, (r) both of SEQ ID NOs: 139 and 140, (s) both of SEQ ID NOs: 147 and 148, or (t) both of SEQ ID NOs: 149 and 150, (u) both of SEQ ID NO: 157 and 158, or (v) both of SEQ ID NO: 159 and 160.

[0091] In an aspect of the invention, the inventive polypeptide can further comprise the constant region of the inventive TCR set forth above. In this regard, the polypeptide can further comprise the amino acid sequence of SEQ ID NO: 67 (WT murine constant region of a chain), SEQ ID NO: 68 (WT murine constant region of P chain), SEQ ID NO: 63 (substituted murine constant region of a chain), SEQ ID NO: 64 (substituted murine constant region of chain), SEQ ID NO: 65 (a chain constant region of cysteine-substituted, LVL- modified TCR); SEQ ID NO: 66 (P chain constant region of cysteine-substituted, LVL- modified TCR); both SEQ ID NOs: 63 and 64, both SEQ ID NOs: 65 and 66, or both SEQ ID NOs: 67 and 68. Preferably, the polypeptide further comprises the amino acid sequences of both of SEQ ID NOs: 63 and 64, both of SEQ ID NO: 65 and 66, or both of SEQ ID NOs: 67 and 68 in combination with any of the CDR regions or variable regions described herein with respect to other aspects of the invention. In an aspect of the invention, one or both of SEQ ID NOs: 63 and 64 of the polypeptide are as defined in any one of Tables 3-5.

[0092] In an aspect of the invention, the inventive polypeptide can comprise the entire length of an a or p chain of the TCR described herein. In this regard, the inventive polypeptide can comprise the amino acid sequence of SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, 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: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ IDNO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ IDNO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ IDNO: 179. SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, or SEQID NO: 184.

[0093] Alternatively, the polypeptide of the invention can comprise both chains of the TCRs described herein. For example, the polypeptide may comprise the amino acid sequences of: both of SEQ ID NOs: 69-70, both of SEQ ID NOs: 71-72, both of SEQ ID NOs: 73-74, both of SEQ ID NOs: 75-76. both of SEQ ID NOs: 77-78. both of SEQ ID NOs: 79-80, both of SEQ ID NOs: 81-82, both of SEQ ID NOs: 83-84, both of SEQ ID NOs: 85-86. both of SEQ ID NOs: 87-88. both of SEQ ID NOs: 161 and 162, both of SEQ ID NOs: 163 and 1 4, both of SEQ ID NOs: 1 5 and 166, both of SEQ ID NOs: 167 and 1 8, both of SEQ ID NOs: 169 and 170, both of SEQ ID NOs: 171 and 172, both of SEQ ID NOs: 173 and 174, both of SEQ ID NOs: 175 and 176, both of SEQ ID NOs: 177 and 178, both of SEQ ID NOs: 179 and 180, (65) both of SEQ ID NOs: 181 and 182, or (66) both of SEQ ID NOs: 183 and 184.

[0094] An aspect of the invention provides a protein comprising at least one of the polypeptides described herein. By "protein" is meant a molecule comprising one or more polypeptide chains.

[0095] In an aspect of the invention, the protein of the invention can comprise (a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 1-3 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 4-6; (b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 11-13 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 14-16; (c) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 21-23 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 24-26; (d) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 31-33 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 34-36; (e) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 41-43 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 44-46; (I) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 101-103 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 104-106; (g) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 111-113 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 114-1 16; (h) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 121-123 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 124- 126; (i) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 131- 133 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 134-136; (j) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 141-143 and a second poly peptide chain comprising the amino acid sequences of SEQ ID NOs: 144-146; or (k) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 151-153 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 154-156.

[0096] In another aspect of the invention, (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: 9 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10; (iii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18; (iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 20; (v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28; (vi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30; (vii) 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; (viii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40; (ix) 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; (x) the first polypeptide chain comprises the ammo acid sequence of SEQ ID NO: 49 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 50; (xi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108; (xii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110; (xiii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 117 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118; (xiv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120; (xv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128; (xvi) 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: 130; (xvii) the first polypeptide chain comprises the amino acidsequence of SEQ ID NO: 137 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138; (xviii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140; (xix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148; (xx) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150; (xxi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158; or (xxii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160.

[0097] The inventive protein may further comprise any of the constant regions described herein with respect to other aspects of the invention. In this regard, in an aspect of the invention, (i) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 63 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 64; (ii) the first polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 65 and the second polypeptide chain may further comprise the amino acid sequence of SEQ ID NO: 66; or (ii) the first polypeptide chain may comprise the amino acid sequence of SEQ ID NO: 67 and the second polypeptide chain may comprise the amino acid sequence of SEQ ID NO: 68. In an aspect of the invention, one or both of SEQ ID NOs: 63 and 64 of the protein are as defined in any one of Tables 3-5.

[0098] The inventive protein may comprise a full length a or [3 chain, as described herein with respect to other aspects of the invention. In this regard, in an aspect of the invention, (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70; (ii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72; (iii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74; (iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76; (v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77 and the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO: 78; (vi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 79 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80; (vii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 81 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82; (viii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 83 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84; (ix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86; (x) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88; (xi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162; (xii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164; (xiii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166; (xiv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168; (xv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170; (xvi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172; (xvii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174; (xviii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176; (xix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178; (xx) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 180; (xxi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182; or (xxii) the firstpolypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184.

[0099] The protein of the invention can be a TCR. Alternatively, if, for example, the protein comprises a single polypeptide chain comprising the amino acid sequences of both the TCR a and [3 chains, or if the first and / or second polypeptide chain(s) of the protein further comprise(s) other amino acid sequences, e.g., an amino acid sequence encoding an immunoglobulin or a portion thereof, then the inventive protein can be a fusion protein. In this regard, the invention also provides a fusion protein comprising at least one of the inventive polypeptides described herein along with at least one other polypeptide. The other polypeptide can exist as a separate polypeptide of the fusion protein, or can exist as a polypeptide, which is expressed in frame (in tandem) with one of the inventive polypeptides described herein. The other polypeptide can encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to an immunoglobulin. CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g.. CDla, CDlb, CDlc. CDld. etc.

[0100] The fusion protein can comprise one or more copies of the inventive polypeptide and / or one or more copies of the other polypeptide. For instance, the fusion protein can comprise 1, 2, 3, 4. 5, or more, copies of the inventive polypeptide and / or of the other polypeptide. Suitable methods of making fusion proteins are known in the art, and include, for example, recombinant methods.

[0101] In some aspects of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the a chain and the (3 chain. In this regard, the TCRs, polypeptides, and proteins of the invention may further comprise a linker peptide. The linker peptide may advantageously facilitate the expression of a recombinant TCR, polypeptide, and / or protein in a host cell. The linker peptide may comprise any suitable amino acid sequence. The linker peptide may be a cleavable linker peptide. For example, the linker peptide may be a furin-SGSG-P2A linker peptide comprising the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 93). Upon expression of the construct including the linker peptide by a host cell, the linker peptide may be cleaved, resulting in separated a and (3 chains. In an aspect of the invention, the TCR, polypeptide, or protein may comprise an amino acid sequence comprising a full-length a chain, a full-length [3 chain, and a linker peptide positioned between the a and [3 chains.

[0102] The protein of the invention can be a recombinant antibody, or an antigen binding portion thereof, comprising at least one of the inventive polypeptides 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 invention and a polypeptide chain of an antibody, or an antigen binding portion thereof. The polypeptide of an antibody, or antigen binding portion thereof, can be a heavy chain, a light chain, a variable or constant region of a heavy or light chain, a single chain variable fragment (scFv), or an Fc, Fab, or F(ab)2' fragment of an antibody, etc. The polypeptide chain of an antibody, or an antigen binding portion thereof, can exist as a separate polypeptide of the recombinant antibody. Alternatively, the polypeptide chain of an antibody, or an antigen binding portion thereof, can exist as a polypeptide, which is expressed in frame (in tandem) with the polypeptide of the invention. The polypeptide of an antibody, or an antigen binding portion thereof, can be a polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0103] Included in the scope of the invention are functional variants of the inventive TCRs, polypeptides, or proteins described herein. The term “functional variant,’’ as used herein, refers to a TCR, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, which functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants encompass, for example, those variants of the TCR, polypeptide, or protein described herein (the parent TCR, polypeptide, or protein) that retain the ability to specifically bind to the G12D RAS, G12V RAS, G13D RAS or Q61R RAS for which the parent TCR has antigenic specificity or to which the parent polypeptide or protein specifically binds, to a similar extent, the same extent, or to a higher extent, as the parent TCR, polypeptide, or protein. In reference to the parent TCR, polypeptide, or protein, the functional variant can, for instance, be at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99% or more identical in amino acid sequence to the parent TCR. polypeptide, or protein, respectively.

[0104] The functional variant can, for example, comprise the amino acid sequence of the parent TCR, polypeptide, or protein with 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 certain physical and / or chemical properties is exchanged for another amino acid that has the same chemical or physical properties. Forinstance, the conservative amino acid substitution can be an acidic amino acid substituted for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Vai, He, Leu, Met, Phe, Pro, Trp, Vai, etc.), a basic amino acid substituted for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain substituted for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc.

[0105] Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferable for the non-conservative amino acid substitution to 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 as compared to the parent TCR, polypeptide, or protein.

[0106] The TCR, polypeptide, or protein can consist essentially of the specified amino acid sequence or sequences described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not materially change the biological activity of the TCR, polypeptide, or protein. In this regard, the inventive TCR, polypeptide, or protein can, for example, consist essentially of the amino acid sequence of SEQ ID NO: 69. SEQ ID NO: 70. SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, 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: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164. SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171 , SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, or SEQ ID NO: 184, both of SEQ ID NOs: 69-70, both of SEQ ID NOs: 71-72, both of SEQ ID NOs: 73-74, both of SEQ ID NOs: 75-76, both of SEQ ID NOs: 77- 78, both of SEQ ID NOs: 79-80, both of SEQ ID NOs: 81-82, both of SEQ ID NOs: 83-84, both of SEQ ID NOs: 85-86, or both of SEQ ID NOs: 87-88, both of SEQ ID NOs: 161 and 162, both of SEQ ID NOs: 163 and 164. both of SEQ ID NOs: 165 and 166, both of SEQ ID NOs: 167 and 168. both of SEQ ID NOs: 169 and 170, both of SEQ ID NOs: 171 and 172. both of SEQ ID NOs: 173 and 174, both of SEQ ID NOs: 175 and 176, both of SEQ ID NOs:177 and 178. both of SEQ ID NOs: 179 and 180, (65) both of SEQ ID NOs: 181 and 182, or (66) both of SEQ ID NOs: 183 and 184.

[0107] Also, for instance, the inventive TCRs, polypeptides, or proteins can consist essentially of the amino acid sequence(s) of (1) SEQ ID NO: 7, (2) SEQ ID NO: 8, (3) SEQ ID NO: 9, (4) SEQ ID NO: 10, (5) SEQ ID NO: 17, (6) SEQ ID NO: 18, (7) SEQ ID NO: 19, (8) SEQ ID NO: 20, (9) SEQ ID NO: 27, (10) SEQ ID NO: 28, (11) SEQ ID NO: 29, (12) SEQ ID NO: 30, (13) SEQ ID NO: 37, (14) SEQ ID NO: 38, (15) SEQ ID NO: 39, (16) SEQ ID NO: 40, (17) SEQ ID NO: 47, (18) SEQ ID NO: 48, (19) SEQ ID NO: 49, (20) SEQ ID NO: 50, (21) SEQ ID NO: 107, (22) SEQ ID NO: 108, (23) SEQ ID NO: 109, (24) SEQ ID NO: 110. (25) SEQ ID NO: 117. (26) SEQ ID NO: 118. (27) SEQ ID NO: 119, (28) SEQ IDNO: 120, (29) SEQ ID NO: 127, (30) SEQ ID NO: 128, (31) SEQ ID NO: 129, (32) SEQ IDNO: 130, (33) SEQ ID NO: 137, (34) SEQ ID NO: 138, (35) SEQ ID NO: 139, (36) SEQ IDNO: 140, (37) SEQ ID NO: 147. (38) SEQ ID NO: 148. (39) SEQ ID NO: 149, (40) SEQ IDNO: 150. (41) SEQ ID NO: 157. (42) SEQ ID NO: 158. (43) SEQ ID NO: 159, (44) SEQ IDNO: 160, (45) both of SEQ ID NOs: 7 and 8, (46) both of SEQ ID NOs: 9 and 10, (47) both of SEQ ID NOs: 17 and 18, (48) both of SEQ ID NOs: 19 and 20, (49) both of SEQ ID NOs: 27 and 28, (50) both of SEQ ID NOs: 29 and 30, (51) both of SEQ ID NOs: 37 and 38, (52) both of SEQ ID NOs: 39 and 40, (53) both of SEQ ID NOs: 47 and 48, (54) both of SEQ ID NOs: 49 and 50, (55) both of SEQ ID NOs: 107 and 108, (56) both of SEQ ID NOs: 109 and 1 10, (57) both of SEQ ID NOs: 117 and 118, (58) both of SEQ ID NOs: 119 and 120, (59) both of SEQ ID NOs: 127 and 128, (60) both of SEQ ID NOs: 129 and 130, (61) both of SEQ ID NOs: 137 and 138, (62) both of SEQ ID NOs: 139 and 140, (63) both of SEQ ID NOs: 147 and 148. (64) both of SEQ ID NOs: 149 and 150. (65) both of SEQ ID NOs: 157 and 158, or (66) both of SEQ ID NOs: 159 and 160. Furthermore, the inventive TCRs, polypeptides, or proteins can consist essentially of the amino acid sequences of (a) all of SEQ ID NOs: 1-3, (b) all of SEQ ID NOs: 4-6, (c) all of SEQ ID NOs: 1-6, (d) all of SEQ ID NOs: 11-13, (e) all of SEQ ID NOs: 14-16, (f) all of SEQ ID NOs: 11-16, (g) all of SEQ ID NOs: 21-23, (h) all of SEQ ID NOs: 24-26, (i) all of SEQ ID NOs: 21-26, (j) all of SEQ ID NOs: 31-33, (k) all of SEQ ID NOs: 34-36, (1) all of SEQ ID NOs: 31-36, (m) all of SEQ ID NOs: 41-43, (n) all of SEQ ID NOs: 44-46, (o) all of SEQ ID NOs: 41-46, (p) all of SEQ ID NOs: 101-103, (q) all of SEQ ID NOs: 104-106, (r) all of SEQ ID NOs: 101-106, (s) all of SEQ ID NOs: 111-113. (t) all of SEQ ID NOs: 114-116. (u) all of SEQ ID NOs: 111-116, (v) all of SEQ ID NOs: 121-123, (w) all of SEQ ID NOs: 124-126, (x) all of SEQ ID NOs: 121-126,(y) all of SEQ ID NOs: 131-133, (z) all of SEQ ID NOs: 134-136, (aa) all of SEQ ID NOs: 131-136, (bb) all of SEQ ID NOs: 141-143, (cc) all of SEQ ID NOs: 144-146, (dd) all of SEQ ID NOs: 141-146, (ee) all of SEQ ID NOs: 151-153, (ff) all of SEQ ID NOs: 154-156, or (gg) all of SEQ ID NOs: 151-156. The TCRs, polypeptides, and proteins of the invention can be of any length, i.e., can comprise any number of amino acids, provided that the TCRs, polypeptides, or proteins retain their biological activity, e.g., the ability to specifically bind to G12D RAS, G12V RAS, G13D RAS or Q61R RAS; detect cancer in a mammal; or treat or prevent cancer in a mammal, etc. For example, the polypeptide can be in the range of from 50 to 5000 amino acids long, such as 50, 70, 75. 100, 125, 150, 175, 200. 300, 400, 500, 600, 700, 800. 900, 1000 or more amino acids in length. In this regard, the polypeptides of the invent on also include oligopeptides.

[0108] The TCRs, polypeptides, and proteins of the invention can comprise 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, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4- chlorophenylalanine. 4-carboxyphenylalanine, p-phenylserine p-hydroxyphenylalanine, phenylglycine, cz-naphthyl alanine, cyclohexylalanine, cyclohexylglycine, indoline-2- carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N’-benzyl-N'-methyl-lysine, N’,N’-dibenzyl-lysine. 6- hydroxylysine. ornithine, cz-aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a-aminocycloheptane carboxylic acid, oc-(2-amino-2-norbomane)-carboxylic acid, oqy-diaminobutyric acid, a,p-diaminopropionic acid, homophenylalanine, and a-tert- butylglycine.

[0109] The TCRs, polypeptides, and proteins of the invention can be glycosylated, amidated, carboxylated, phosphory lated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and / or optionally dimerized or polymerized, or conjugated.

[0110] The inventive TCRs, polypeptides, and proteins described herein (including any of the functional portions or variants thereof) are also contemplated to be useful as the soluble TCR component of bispecific engager TCR fusion proteins (e.g., IMMTAC (immune- mobilizing monoclonal TCRs against cancer) molecules). Bispecific engager TCR fusionproteins have two components. One component comprises a soluble TCR. The other component comprises an anti-CD3 effector. The anti-CD3 effector may be any molecule that engages with a CD3 molecule on T cells and activates a T cell immune response. For example, the anti-CD3 effector may be an anti-CD3 antibody or anti-CD3 antibody fragment. The soluble TCR component of the bispecific engager TCR fusion protein binds to the target antigen presented on the surface of cancer cells presented by an HLA molecule. The anti- CD3 effector component engages a CD3 molecule on T cells. The engagement of these components of the bispecific engager TCR fusion protein triggers the activation and recruitment of T cells and redirects T-cell killing to tumor cells. An aspect of the invention provides a bispecific engager TCR fusion protein comprising (i) any of the inventive TCRs, polypeptides, or proteins (including any of the functional portions or variants thereof) described herein and (ii) an anti-CD3 effector. Hereinafter, references to "protein(s)" also encompass the bispecific engager TCR fusion proteins described herein, unless specified otherwise.[01H] The TCR, polypeptide, and / or protein of the invention can be obtained by methods known in the art such as, for example, de novo synthesis. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Green and Sambrook, Molecular Cloning: A Laboratory Manual. 4thed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, the TCRs, polypeptides, and / or proteins described herein can be synthesized by any of a variety of commercial entities. In this respect, the inventive TCRs, polypeptides, and proteins can be synthetic, recombinant, isolated, and / or purified. An aspect 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 aspect of the invention provides an isolated or purified TCR, polypeptide, or protein that results from expression of any of the nucleic acids or vectors described herein in a cell. Still another aspect of the invention provides a method of producing any of the TCRs, polypeptides, or proteins described herein, the method comprising culturing any of the host cells or populations of host cells described herein so that the TCR, polypeptide, or protein is produced.

[0112] Included in the scope of the invention are conjugates, e.g.. bioconjugates, comprising any of the inventive TCRs, polypeptides, or proteins (including any of the functional portions or variants thereof), nucleic acids, recombinant expression vectors, hostcells, or populations of host cells. Conjugates, as well as methods of synthesizing conjugates in general, are known in the art.

[0113] An aspect of the invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs. polypeptides, or proteins described herein. "Nucleic acid," as used herein, includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered intemucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide. In an aspect, the nucleic acid comprises complementary DNA (cDNA). It is generally preferred that the nucleic acid does not comprise any insertions, deletions, inversions, and / or substitutions. However, it may be suitable in some instances, as discussed herein, for the nucleic acid to comprise one or more insertions, deletions, inversions, and / or substitutions.

[0114] Preferably, the nucleic acids of the invention are recombinant. As used herein, the term "recombinant" refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0115] The nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, for example, Green and Sambrook et al., supra. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides). Examples of modified nucleotides that can be used to generate the nucleic acids include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5 -chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl- 2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, P-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2- methyl adenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-substituted adenine, 7-methylguanine. 5-methylaminomethyluracil. 5-methoxyaminomethyl-2-thiouracil, P-D-mannosylqueosine, 5'-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v). wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the invention could be prepared by any of a variety of commercial entities.

[0116] The nucleic acid can comprise any nucleotide sequence which encodes any of the TCRs, polypeptides, or proteins described herein. In an aspect of the invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound to any particular theory or mechanism, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

[0117] The invention also provides a nucleic acid comprising a nucleotide sequence which is complementary to the nucleotide sequence of any of the nucleic acids described herein or a nucleotide sequence which hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0118] The nucleotide sequence which hybridizes under stringent conditions preferably hybridizes under high stringency conditions. By “high stringency conditions” is meant that the nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any of the nucleic acids described herein) in an amount that is detectably stronger than non-specific hybridization. High stringency conditions include conditions which would distinguish a polynucleotide with an exact complementary sequence, or one containing only a few scattered mismatches from a random sequence that happened to have a few small regions (e.g., 3-10 bases) that matched the nucleotide sequence. Such small regions of complementarity are more easily melted than a full-length complement of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and / or high temperature conditions, such as provided by 0.02-0. 1 M NaCl or the equivalent, at temperatures of 50-70 °C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularly suitable for detecting expression of any of the inventive TCRs. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide.

[0119] An aspect of the invention also provides a nucleic acid comprising a nucleotide sequence that is at least 80% or more, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid may consist essentially of any of the nucleotide sequences described herein.

[0120] An aspect of the invention provides 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 sequence, respectively, encode the amino sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 17 and 18; 18 and 17; 19 and 20; 20 and 19; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 37 and 38; 38 and 37; 39 and 40; 40 and 39; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 69 and 70; 70 and 69; 71 and 72; 72 and 71; 73 and 74; 74 and 73; 75 and 76; 76 and 75; 77 and 78; 78 and 77; 79 and 80; 80 and 79; 81 and 82; 82 and 81; 83 and 84; 84 and 83; 85 and 86; 86 and 85; 87 and 88; or 88 and 87; 107 and 108; 108 and 107; 109 and 110; 110 and 109; 117 and 118; 118 and 117; 119 and 120;120 and 119; 127 and 128; 128 and 127; 129 and 130; 130 and 129; 137 and 138; 138 and137; 139 and 140; 140 and 139; 147 and 148; 148 and 147; 149 and 150; 150 and 149; 157 and 158; 158 and 157; 159 and 160; 160 and 159; 161 and 162; 162 and 161; 163 and 164;164 and 163; 165 and 166; 166 and 165; 167 and 168; 168 and 167; 169 and 170; 170 and169; 171 and 172; 172 and 171; 173 and 174; 174 and 173; 175 and 176; 176 and 175; 177 and 178; 178 and 177; 179 and 180; 180 and 179; 181 and 182; 182 and 181; 183 and 184; or 184 and 183.

[0121] In an aspect of the invention, the isolated or purified nucleic acid further comprises a third nucleotide sequence interposed between the first and second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide. In an aspect of the invention, the cleavable linker peptide comprises the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 93).

[0122] The nucleic acids of the invention can be incorporated into a recombinant expression vector. In this regard, the invention provides a recombinant expression vector comprising any of the nucleic acids of the invention. In an aspect of the invention, the recombinant expression vector comprises a nucleotide sequence encoding the a chain, the [3 chain, and linker peptide.

[0123] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring. The inventive recombinant expression vectors can comprise any type of nucleotide, including, but not limited to DNA and RNA, which can be single-stranded or double-stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally-occurring, non- naturally-occurring intemucleotide linkages, or both types of linkages. Preferably, the non- naturally occurring or altered nucleotides or intemucleotide linkages do not hinder the transcription or replication of the vector.

[0124] The recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla. CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as LGT10, ZGTI 1, XZapII (Stratagene), XEMBL4, and XNM1149, also can be used. Examples of plant expression vectors include pBIOl. 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, e.g., a retroviral vector. In an especially preferred aspect, the recombinant expression vector is an MSGV1 vector. In an aspect of the invention, the recombinant expression vector is a transposon, a retroviral vector, or a lentiviral vector.

[0125] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green and Sambrook et al., supra. Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replicationsystems can be derived, e.g.. from ColEl, 2 p plasmid, , SV40. bovine papillomavirus, and the like.

[0126] Desirably, the recombinant expression vector comprises regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA- based.

[0127] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g.. resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host cell to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0128] The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the TCR, polypeptide, or protein, or to the nucleotide sequence which is complementary7to or which hybridizes to the nucleotide sequence encoding the TCR. polypeptide, or protein. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a non-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.

[0129] The inventive recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0130] Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosinedeaminase, purine nucleoside phosphorylase, nitroreductase, and the inducible caspase 9 gene system.

[0131] Another aspect of the invention further provides a host cell comprising any of the nucleic acids or recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance. DH5a E. coli cells. Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., a DH5a 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. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell preferably is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell. In an aspect of the invention, the host cell is a human lymphocyte. In another aspect of the invention, the host cell is selected from the group consisting of a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a natural killer (NK) cell, a macrophage, a pluripotent cell, and a multipotent cell. Still another aspect of the invention provides a method of producing a host cell expressing a TCR that has antigenic specificity for the peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89), MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91), MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95), VVGAVGVGK (SEQ ID NO: 97), or VVVGAVGVGK (SEQ ID NO: 99). the method comprising contacting a cell with any of the vectors described herein under conditions that allow introduction of the vector into the cell. Such a method may be carried out in vitro or in vivo.

[0132] For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl. etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. Preferably, the T cell isa human T cell. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+double positive T cells, CD4+helper T cells, e.g., Thi and Th2 cells, CD4+T cells, CD8+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.

[0133] In an aspect of the invention, the host cell is a pluripotent cell or a multipotent cell. Pluripotent cells have the capacity to give rise to any of the three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent cells may comprise, for example, stem cells, e.g., embryonic stem cells, nuclear transfer derived embryonic stem cells, induced pluripotent stem cells (iPSC), etc. Multipotent cells may comprise, for example, hematopoietic stem cells. Modifying, e.g., reprogramming, cells to a pluripotent state refers to the reversion of a cell to a pluripotent cell and is described for example, in Crompton et al., Trends Immunol., 35(4): 178-185 (2014). Exemplary techniques may include somatic cell nuclear transfer (SCNT). cell-cell fusion, and direct reprogramming. Examples of methods for carrying out cell-cell fusion are described, for example, in Ogle et al., Nat. Rev. Mol. Cell Biol. 6: 567-75 (2005) and Zhou et al., Cell Stem Cell, 3: 382-388 (2008). Examples of methods for carry ing out SCNT are described, for example, in Hanna et al., Cell, 143: 508- 525 (2010); Stadtfeld et al., Genes Dev.. 24: 2239-2263 (2010); Wilmut et al., Nature, 385: 810-813 (1997); Vizcardo et al.. Cell Stem Cell. 12: 31-36 (2013); and Crompton et al., Cell Stem Cell, 12: 6-8 (2013). In an aspect of the invention, the host cell is an iPSC that was prepared by reprogramming, any of the host cells described herein (e.g., T cells, NK cells, or invariant natural killer T cells) to a pluripotent state.

[0134] Also provided by the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the recombinant expression vectors described, in addition to at least one other cell, e.g., a host cell (e.g., a T cell), which does not comprise any of the recombinant expression vectors, or a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an ery throcyte, a hepatocyte, an endothelial cell, an epithelial cells, a muscle cell, a brain cell, etc. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly of host cells (e.g., consisting essentially ol) comprising the recombinant expression vector. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising a recombinant expression vector, such that all cells of the populationcomprise the recombinant expression vector. In one aspect of the invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0135] In an aspect of the invention, the numbers of cells in the population may be rapidly expanded. Expansion of the numbers of T cells can be accomplished by any of a number of methods as are known in the art as described in, for example, U.S. Patent 8,034,334; U.S. Patent 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 an aspect, expansion of the numbers of T cells is carried out by culturing the T cells with OKT3 antibody. IL-2, and feeder PBMC (e.g., irradiated allogeneic PBMC).

[0136] The inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof), can be isolated and / or purified. The term "isolated," as used herein, means having been removed from its natural environment. The term "purified," as used herein, means having been increased in purity, wherein "purity" is a relative term, and not to be necessarily construed as absolute purity. For example, the purity can be at least 50%, can be greater than 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. or can be 100%.

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

[0138] Preferably, the carrier is a pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used for theparticular inventive TCR material under consideration. Methods for preparing administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, 23rd Ed., Pharmaceutical Press (2020). It is preferred that the pharmaceutically acceptable carrier be one which has no detrimental side effects or toxicity’ under the conditions of use.

[0139] The choice of carrier will be determined in part by the particular inventive TCR material, as well as by the particular method used to administer the inventive TCR material. Accordingly, there are a variety7of suitable formulations of the pharmaceutical composition of the invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or interperitoneal administration. More than one route can be used to administer the inventive TCR materials, and in certain instances, a particular route can provide a more immediate and more effective response than another route.

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

[0141] For purposes of the invention, the amount or dose (e.g., numbers of cells when the inventive TCR material is one or more cells) of the inventive TCR material administered should be sufficient to effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a reasonable time frame. For example, the dose of the inventive TCR material should be sufficient to bind to a cancer antigen (e.g., G12D RAS, G12V RAS, G13D RAS or Q61R RAS), or detect, treat or prevent cancer in a period of from 2 hours or longer, e.g.. 12 to 24 or more hours, from the time of administration. In certain aspects, the time period could be even longer. The dose will be determined by the efficacy of the particular inventive TCR material and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0142] Many assays for determining an administered dose are known in the art. For purposes of the invention, an assay, which comprises comparing the extent to which targetcells are lysed or IFN-y is secreted by T cells expressing the inventive TCR, polypeptide, or protein upon administration of a given dose of such T cells to a mammal among a set of mammals of which each is given a different dose of the T cells, could be used to determine a starting dose to be administered to a mammal. The extent to which target cells are lysed or IFN-y is secreted upon administration of a certain dose can be assayed by methods known in the art.

[0143] The dose of the inventive TCR material also will be determined by the existence, nature and extent of any adverse side effects that might accompany the administration of a particular inventive TCR material. Typically, the attending physician will decide the dosage of the inventive TCR material with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inventive TCR material to be administered, route of administration, and the severity of the cancer being treated. In an aspect in which the inventive TCR material is a population of cells, the number of cells administered per infusion may vary, e.g., from 1 x 106to 1 x 1012cells or more. In certain aspects, fewer than 1 x 106cells may be administered.

[0144] One of ordinary skill in the art will readily appreciate that the inventive TCR materials of the invention can be modified in any number of ways, such that the therapeutic or prophylactic efficacy of the inventive TCR materials is increased through the modification. For instance, the inventive TCR materials can be conjugated either directly or indirectly through a bridge to a chemotherapeutic agent. The practice of conjugating compounds to a chemotherapeutic agent is known in the art. One of ordinary skill in the art recognizes that sites on the inventive TCR materials, which are not necessary for the function of the inventive TCR materials, are suitable sites for attaching a bridge and / or a chemotherapeutic agent, provided that the bridge and / or chemotherapeutic agent, once attached to the inventive TCR materials, do(es) not interfere with the function of the inventive TCR materials, i.e., the ability to bind to G12D RAS, G12V RAS. G13D RAS or Q61R RAS or to detect, treat, or prevent cancer.

[0145] It is contemplated that the inventive pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, and populations of cells can be used in methods of treating or preventing cancer. Without being bound to a particular theory, the inventive TCRs are believed to bind specifically to G12D RAS, G12V RAS, G13D RAS or Q61R RAS, such that the TCR (or related inventive polypeptide or protein), when expressed by a cell, is able to mediate an immune responseagainst a target cell expressing G12D RAS, G12V RAS, G13D RAS or Q61R RAS, respectively. In this regard, an aspect of 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, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0146] An aspect of the invention provides a method of inducing an immune response against a 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 which encodes any of the TCRs, polypeptides, or proteins described herein, in an amount effective to induce an immune response against the cancer in the mammal.

[0147] An aspect of the invention provides 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, proteins described herein, or any host cell or population of cells comprising a recombinant vector which encodes any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0148] An aspect of the invention provides 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 which encodes any of the TCRs. polypeptides, or proteins described herein, for use in inducing an immune response against a cancer in a mammal.

[0149] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment or prevention of cancer in a mammal.Furthermore, the treatment or prevention provided by the inventive method 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 the regression of a tumor. Also, for purposes herein, "prevention" can encompass delaying the onset of the cancer, or a symptom or condition thereof. Alternatively or additionally, “prevention” may encompass preventing or delaying the recurrence of cancer, or a symptom or condition thereof.

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

[0151] With respect to the inventive method of detecting cancer in a mammal, the sample of cells can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein fraction, or a nucleic acid fraction.

[0152] For purposes of the inventive method of detecting cancer, the contacting can take place in vitro or in vivo with respect to the mammal. Preferably, the contacting is in vitro.

[0153] Also, detection of the complex can occur through any number of ways known in the art. For instance, the inventive TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells, described herein, can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and element particles (e.g., gold particles).

[0154] For purposes of the inventive methods, wherein host cells or populations of cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0155] With respect to the inventive methods, the cancer can be any cancer, including any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, 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 ofthe vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, uterine cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypophary nx cancer, kidney cancer, lary nx cancer, leukemia, liver cancer, lung cancer, malignant mesothelioma, melanoma, mucinous adenocarcinoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, ureter cancer, and urinary’ bladder cancer. A preferred cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, thyroid cnacer, leukemia, melanoma, or prostate cancer. Preferably, the lung cancer is lung adenocarcinoma, the ovarian cancer is epithelial ovarian cancer, the thyroid cancer is follicular thyroid cancer, the colorectal cancer is colorectal carcinoma, the melanoma is cutaneous melanoma, and the pancreatic cancer is pancreatic adenocarcinoma.

[0156] In an aspect of the invention, the cancer expresses a mutated human RAS amino acid sequence with a one or both of (i) glycine at position 12 with aspartic acid, (ii) glycine at position 12 with valine, (iiii) glycine at position 13 with aspartic acid or (iv) glutamine at position 61 with arginine, wherein the mutated human RAS amino acid sequence is a mutated human KRAS, a mutated human HRAS. or a mutated human NRAS ammo acid sequence, and wherein positions 12, 13 and 61 are defined by reference to the WT human KRAS, WT human HRAS, or WT human NRAS protein, respectively. The mutated human KRAS, mutated human HRAS, and mutated human NRAS expressed by the cancer may be as described herein with respect to other aspects of the invention.

[0157] The mammal referred to in the inventive methods can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

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

[0159] The following materials and methods were employed with respect to the experiments described in Examples 1-23.MethodsPatient Demographics

[0160] The demographics of Patients 4342 and 4400 are shown in Table 6.TABLE 6Tumor Samples

[0161] All tumor samples and patient-derived blood products used in the generation of lymphocytes and antigen presenting cells were obtained from patients enrolled in NCT00068003 and NCT00001823 clinical protocols at the National Institutes of Health (NIH). Both protocols received approval from the Institutional Review Board (IRB) of theNational Cancer Institute (NCI) at the NIH. Informed consent was obtained and documented in accordance with the Declaration of Helsinki. Enrollment criteria required all patients to be 18 years of age or older with stage IV metastatic epithelial cancers with measurable metastatic disease and Eastern Cooperative Oncology Group (ECOG) score 0 or 1. All patients were required to be free of all systemic infections at time of sample procurement and treatment. At the time of metastasectomy, all patients were required to abstain from systemic treatment for a minimum of one month prior to procedure. Patients were allowed to have previously participated in any therapeutic strategy (surgery7, chemotherapy, radiotherapy, immunotherapy, etc.), however they could not have any history of grade 3 or 4 adverse side effects from immunotherapy options (Anti-PD-1, Anti-PD-Ll, Anti-CTLA-4) which would preclude their administration with trial therapy. All tumor samples were processed with a combination of mechanical separation, enzy matic digestion and finally GENTLEMACS dissociation technology (Miltenyi Biotec, Gaithersburg. MD) as previously described (Lowery et al., Science, 375(6583): 877-884 (2022)). Resulting single cell suspensions were cryopreserved within NCI tissue bank until time of experimentation. At time of intervention, samples were thawed and rested for approximately 24 hours in T-cell media containing DNAse (Genentech, San Francisco, CA) without introduction of cytokines. For all patient samples, Ficoll-Hypaque gradient (LSM; ICN Biomedicals. Costa Mesa, CA) was used to isolate lymphocytes from resultant cellular debris from tumor processing protocol.Antibodies and cell sorting

[0162] All fluorescently labeled antibodies used for cell sorting were purchased from BD Bioscience (Franklin Lakes, NJ) and Biolegend (San Diego, CA). For initial sorting of three historic samples (4342, 4393, 4283), single cell sequencing fluorescent labels included CD3 (SK7), CD4 (SK3), and CD8(SK1). Following fluorescent antibody labeling, samples underwent cellular indexing of transcriptomes and epitopes (CITE-Seq) via labeling with TOTALSEQ-C Human Universal Cocktail (Biolegend) containing antibodies reactive to 130 unique cell surface proteins.

[0163] On repeat single cell sorting of patient sample 4342 for investigation of single marker performance, cells were labelled collectively with CD3 (SK7), CD4 (SK3). and CD8(RPA-T8) followed by separate patient staining with ADGRG-1 (CG4), CD 103 (Ber- ACT8), CD25 (BC96), CD146 (P1H12), CD196 (G034E3), CD39 (Al), CD57 (QA17A04),PD-1 (EH12.2H7). CD86 (IT2.2), CD278 (C398.4A) and TIM3 (F38-2E2) (BD Bioscience, Biolegend).

[0164] On repeat single cell sorting of 4393 and for all prospective patient samples (4400, 4523, 4432, 4520, 4560), patients underwent labeling with universal cocktail containing CD4 (SK3), CD3 (SK7), CD8 (RPA-T8), PD-1 (EH12.2H7). CD278 (C398.4A), ADGRG-1 (CG4), CD86 (IT2.2), CD57 (QA17A04), CD39 (Al), TIM-3 (F38-2E2) (BD Bioscience, Biolegend).

[0165] Cells were sorted via flow cytometry via Sony MA900 Multi-Application Cell Sorter (Sony Biotechnology. San Jose, CA). Mean fluorescent intensity (MFI) gates for each population were determined according to isotype controls for each individual patient sample.Single-cell sorting and transcriptomic analysis

[0166] Following cell sorting, single cell sequencing was performed on the original three samples (4283, 4342, 4393) via bulk CD4+ and CD8+ TIL populations. Their single-cell gene expression libraries were sequenced with aNEXTSEQ 550 sequencer with output processed using the Cell Ranger V.3.0 pipeline (lOx Genomics. Pleasanton, CA) and analysis completed with R package Seurat V.4 as previously described (Chatani et al., J. Immunother. Cancer, 11(5): e006264 (2023); Krishna et al.. Science, 370(6522): 1328-1334 (2020);Lowery et al., Science, 375(6583): 877-884 (2022)).T-cell receptor sequencing

[0167] For subsequent sorting performed on 4342, 4393, 4520, 4513, 4400, 4432 and 4560, individual cells were sorted into either 96- or 384- well plates based on expression of individual markers of interest (ADGRG-1, CD39. CD103, CD25. PD-1, CD86, CD57, TIM- 3, CD146, CD196) or combinations (PD-l / ICOS, ADGRG-1 / CD86, ADGRG-l / PD-1, ADGRG-1 / CD86 / PD1). The 96- or 384- well plates underwent TCRot / p pair sequencing.T-cell receptor reconstruction, cloning into expression vectors and expression analysis.

[0168] Upon review of TCRa / p pair sequencing, expanded clones with equivalent a and P pair reads were compiled. Of these expanded clones, the median read counts for both a and P sequences were annotated. In addition to all expanded clones, singly occurring clones expressing equivalent a / p pairs above the previously determined median read count thresholdwere included for candidate TCR constructs. Any 0 sequence containing more than two unique a sequences was discounted. Full length TRAV and TRBV regions were reconstructed using the IMGT database and a modified murine TRAC and TRBC sequence was used for improved stability and to avoid mismatch pairing with endogenously expressed human TCR upon transduction into human peripheral blood T-cells (Chatani et al.. J. Immunother. Cancer, 11(5): e006264 (2023)). The full-length TCR sequence was aligned within a single mRNA with a 2A peptide (RAKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 93) between the TCRa and TCR0 chains and subcloned into the pMSGVl vector by Genscript (Piscataway, NJ) (Chatani et al., J. Immunother. Cancer. 11(5): e006264 (2023)).

[0169] Constructed TCR plasmids, along with pRDl 14 packaging plasmid, were transfected into 293GP human epithelial kidney cells via use of LIPOFECTAMINE 2000 transfection reagent (ThermoFischer Scientific, Waltham, MA). This resulted in generation of retroviral supernatant accumulated over 72 hours. Supernatant was harvested and transfected into healthy donor peripheral blood lymphocytes which were previously stimulated within T-cell media containing anti-CD3 (OKT3, 50ng / mL) and IL-2 (300IU / ml). Transduction was performed via spinoculation as previously described (Chatani et al., J. Immunother. Cancer, 11(5): e006264 (2023), Yang et al., J. Immunother ., 31: 830-9 (2008); Morgan et al., Science. 314: 126-9 (2006)). TCR transduction was confirmed via flow cytometry performed at least seven days post-transduction as well as at the time of reactivity analysis as described in following section.Tcell receptor functional assays

[0170] Transduced TCRs were evaluated for functional reactivity via overnight coculture. Transduced T cells were incubated overnight within enzyme-linked immunospot (ELISpot) plates along with patient-derived antigen presenting cells pulsed with patient specific mutated peptides constructed based on tumor sequencing (Lowery et al., Science, 375(6583): 877-884 (2022)). Following overnight co-culture, cells were stained with fluorescently labeled antibodies (CD3, CD8, CD4, CD137, murine TCR0) (BD Biosciences). ELISpot plates underwent development to detect interferon gamma (IFN-y) release while fluorescently stained T-cells were analyzed via flow' cytometry for upregulation of CD137 (4- 1BB). Positive results were determined by IFN-y release or CD137 upregulation twicebackground as determined by control wells containing T-cells, APCs and DMSO alone. Flow cytometry data was analyzed via FLOWJO software (Tree Star, Ashland, OR).Patient-derived xenograft, organoid and tumor cell line preparation and functional screening

[0171] All patient-derived xenograft (PDX) tumors were implanted into mice approved under the NIH IRB animal protocol number SB 194. Fresh patient tumor samples were mechanically separated into fragments from the initial tissue harvest and implanted subcutaneously at the flank of NOD-scid-IL2rgnull (NSG, NCI, Frederic, MA) mice using a 20-gauge needle. Tumor growth was monitored by investigators weekly. PDX tumors were harvested when their sizes were greater than 1 cm in dimension. All animal use for this study was approved by the NCI Animal Care and Use Committee.

[0172] Simultaneously, fragments from initial tumor resection were processed via combination of mechanical, enzymatic, and finally GENTLEMACS dissociation technology (Miltenyi Biotec) for isolation of tumor cells. Malignant cells were grown within a gel construct in histology-specific cell media to encourage cellular growth and proliferation in the construction of the patient derived organoid.

[0173] Freshly harvested PDX tumor, PDX derived cell lines and organoid derived cell lines were mechanically separated and resuspended in deionized water. Cells underwent flash freeze thaw7process via liquid nitrogen submersion for five iterations. Cells w ere then centrifuged at 16,000g for 20 minutes to complete cell lysis. Following centrifugation, cell lysate mixture underwent mechanical filtration with 100 micron filter. Cell lysate was pulsed overnight onto patient specific antigen presenting cells for use in functional assays.Index sorting analysis

[0174] At time of plate sorting for samples (4393, 4400, 4432, 4520, 4513, 4560), index sorting data was stored on Sony MA900 Multi-Application Cell Sorter (Sony Biosciences). As mentioned previously, MFI gate positioning was determined by isoty pe controls for each individual patient sample. At time of analysis, MFI gate position was recorded and applied to index data for each plate w ell containing a viably sequenced TCR, as described in previous section, to record total cell surface protein expression makeup of cells of interest.Statistical analysis

[0175] Statistical calculations were performed via Prism program v8.0 (GraphPad Software, La Jolla, CA).EXAMPLE 1

[0176] This example demonstrates the transcriptional states of neoantigen-specific CD4+ TIL in freshly resected tumor samples.

[0177] Three retrospective epithelial tumor samples (4342, 4393, 4283) were selected based on availability of cryopreserved tumor digests as well as library' of previously- identified anti-tumor reactive CD4+ TCRs described in a previous study (Lowery et al., Science, 375(6583): 877-884 (2022)). Samples were prepared for sorting and single cell sequencing as described in the methods section. Following single cell RNA-sequencing, readout was processed via established lOx pipeline. Cells were indexed for CD4+ expression and uniform manifold approximation and projection analysis (UMAP) was conducted based on single cell transcriptomic states. Fold change of distinct RNA expression was analyzed. Key TIL-dysfunction genes previously described were plotted (Lowery- et al., Science, 375(6583): 877-884 (2022)). As previously reported, cell states ranged from less differentiated CD4+ TIL states, regulatory TIL states, CD4+ TIL with markers of dysfunction and exhaustion, and possible cytotoxic CD4+ TIL state. In particular, clusters 4. 8 and 9 had high transcriptomic expression of CXCL13, ENTPD1, PDCD1, TIGIT and HAVCR2, which likely- corresponded to anti-tumor reactive CD4+ T-cells (Lowery- et al., Science, 375(6583): 877-884 (2022); Oliveira et al., Nature, 605(7910): 532-538 (2022); Hanada et al., Cancer Cell, 40(5): 479-493 (2022)). Additionally, these three clusters (4, 8 and 9) were found to have lower expression of FOXP3, suggesting that these are tumor-infiltrating dysfunctional TIL, but not T regulatory- CD4+ cells (TREG).

[0178] A library- of 20 previously discovered antitumor, neoantigen-specific T cell receptor clonotypes (NeoCD4+ TCRs) from the three samples was then utilized to locate them within the transcriptional space. In this library-, six TCR clonotypes from 4342, eight from 4283 and two from 4393 were present within the samples. A total of 96 T cells expressing neoantigen-reactive TCRs were present within the samples, with 36 found from 4342, 54 from 4283 and 6 from 4393 consisting of 37.5%, 56.3% and 6.3% of the total clonotypes, respectively. By back-projecting these TCR sequences onto the UMAP, it wasfound that they were largely concentrated within the previously identified candidate clusters (4, 8 and 9). Cluster 4 contained 52.1 % of all present NeoCD4+ clonotypes, followed by cluster 9 (25.0%) then cluster 8 (19.8%). The next nearest cluster (cluster 11) contained only 2.1% of all present NeoCD4+ TCRs. Amongst all cells within individual clusters, cluster 4 comprised 9.35% NeoCD4+ cells, followed by cluster 9 (7.1%) and cluster 8 (5.5%). Notably, cluster 4 also contained the highest number of unique NeoCD4+ TCRs with 12 different Neo-TCRs. Clusters 8 and 9 both contained eight unique Neo-TCR clonotypes. Additionally, a prior NeoCD4 RNA gene signature was projected onto thr cluster analysis, confirming higher gene score within the clusters of interest (4, 8 and 9) (Lowery et al., Science, 375(6583): 877-884 (2022)). A previously published TREG RNA gene signature was included, which showed ambiguity across clusters, with the highest score found within cluster 0 (Wu et al., Nature, (7798): 274-278 (2020)). Thus, bona-fide CD4+ neoantigen- specific antitumor TIL were captured in distinct transcriptional states, as previously reported in these three patient TIL samples.EXAMPLE 2

[0179] This example demonstrates an unbiased definition of the cell surface proteome of CD4+ neoantigen TIL in epithelial cancers.

[0180] To analyze CITEseq data from the same samples, the cell surface protein expression of 137 immune cell surface DNA-barcoded antibodies were then examined. Several candidate markers were found that were shared across two or more of the transcriptional states comprising known neoantigen-specific TIL. Most cell surface proteins corresponded with their consistent transcriptional states. It was then noted that neoantigen CD4+ TIL clusters of interest expressed cell surface protein expression of ADGRG-1, CD86, CD57 and PD-1. Additional markers of CD8+ anti -tumor T-cell reactivity showed no apparent specificity for transcriptional states enriched with CD4+ neoantigen TIL, with CD39 cell surface expression largely comprising Treg CD4+ TIL (Chatani et al., J. Immunother. Cancer, 11(5): e006264 (2023); Lowery et al., Science, 375(6583): 877-884 (2022)). These results show that CD4+ neoantigen TIL had unique cell surface profiles that do not perfectly align with that of CD8+ neoantigen TIL states.EXAMPLE 3

[0181] This example demonstrates the assessment of single cell surface protein markers for enrichment of NeoCD4+ TIL.

[0182] To functionally investigate the single marker performance in enriching for NeoCD4+ T-cells from tumor samples, patient samples 4393 and 4342 were selected from the CITEseq data for additional sorting based on availability of cryopreserved tumor specimens. These samples were prepared as described in the methods section. Sorting revealed population variability between samples, with CD57 containing the largest populations with a median of 8.7% of CD4+ T-cells, followed by PD-1 (8.6%), ADGRG-1 (5.5%), CD39 (4.3%), TIM-3 (4.2%) and CD86 (3.7%).

[0183] The initial RNA-transcriptomic UMAP was used to select several candidate CD4+ TCRs from the NeoTCR4 TIL states (clusters 4. 8 and 9) for neoantigen-assessment by interferon-gamma ELISpot and flow cytometry. Additionally, candidate TCRs were selected from the 4393 surface marker plate sort as per the methods section. A total of 97 candidate TCRs, 28 from 4342 and 69 from 4393, were selected and screened as described in the methods section. Fifteen TCRs from 4342 and fourteen from 4393 screened positive for reactivity against patient specific pooled mutated peptides. Screened positive TCRs underwent repeat co-culture with individual mutated peptides with identification of neoantigen reactivities demonstrated by IFN-y release (ELISpot) and CD137 (4-1BB) upregulation (via flow cytometry).

[0184] Across single marker sorting for historic samples (4342. 4393), ADGRG-1 and PD-1 identified the mostNeoCD4+ T-cells. This was followed by CD86 (10), CD39 (9), CD4 bulk (8), CD57 (7) and TIM-3 (5). PD-1 sorted wells contained the largest number of unique TCRs (13) followed by ADGRG-1 (10), CD39 (8), CD4 bulk (7), CD57 and CD86 (5) and TIM-3 (4). Of note, the majority of anti-tumor reactive CD4+ T-cells were found as single cells compared to expanded clones on an individual plate population. Amongst plate wells containing a productive TCR sequence, as outlined in the methods section, ADGRG-1 contained the highest median percentage of enrichment for NeoCD4+ T-cells with 24.7%, followed by PD-1 (20.8%), CD39 (13.8%), CD86 (12.2%), CD4 bulk (10.6%). CD57 (8.9%) and TIM-3 (7.1%).EXAMPLE 4

[0185] This example demonstrates prospective utilization of single cell surface protein markers for enrichment of NeoCD4+ TIL.

[0186] To prospectively evaluate and validate candidate cell surface marker performance to enrich CD4+ neoantigen TIL, five patient samples were selected for single cell sorting and TCR sequencing (4400, 4432, 4513, 4520 and 4560). Samples were selected based on availability of cryopreserved tumor digest without consideration of CD4+ T-cell reactivity within samples. Patients had varying histology, including colorectal (4400, 4513 and 4432), pancreas (4520) and esophageal cancer (4560). Samples were prepared as described in the methods section.

[0187] Population size of each individual marker again varied across samples. The largest population amongst samples was CD57 with 4.2% of CD4+ cells, then ADGRG-1 (2.9%), CD86 (2.0%), PD-l / ICOS (1.5%) and TIM-3 (1.0%). Following TCR sequencing and analysis as previously described, 261 TCR constructs were screened from 4400 (54), 4520 (44), 4513 (63), 4432 (66) and 4560 (34). Across all samples, NeoCD4+ TCRs were found within 4400 (9) and 4520 (6). Screened positive TCRs were confirmed with repeat coculture against individual long peptides followed by mutated wild type peptide specificity titrations. Screening included cell lysates of patient-derived xenografts, xenograft derived cells lines, and organoids pulsed onto antigen presenting cells as described in the methods section.

[0188] Across the validation samples. CD86 was the most successful in enriching NeoCD4+ T-cells, with 11.1% of sorted wells containing an anti -tumor reactive CD4+ cell, followed by ADGRG-1 (7.4%), CD57 (6.8%), TIM-3 (5.3%) and finally PD-l / ICOS (3.5%). Amongst the two samples with CD4+ reactivity, however, the median cell enrichments were 27.6% (CD86), 18.6% (ADGRG-1). 17.4% (PD-l / ICOS), 13.6% (CD57) and 7.9% (TIM-3) in validation samples. Taken together, these data validate the utility of candidate cell surface markers for the identification and enrichment of CD4+ neoantigen TIL from solid epithelial tumors.EXAMPLE 5

[0189] This example demonstrates a comparison of cell surface protein markers for enrichment of NeoCD4+ TIL across samples.

[0190] To further compare performance of the markers against current published signatures (Duhen et al., J. Clin. Invest., 132(12): el 56821 (2022)), a repeat sort of 4393 was performed to include PD-l / ICOS populations within the analysis. A comprehensive analysis of FACS-index sorting data was then performed to identify the efficient capture of the identified neoantigen-specific CD4+ TIL clonotypes and their corresponding overlap in cell surface expression in various populations. All cells staining above the MFI were identified, consistent with expression of the cell surface marker. Including this data, it was found that amongst all identified NeoCD4+ T-cells, CD86 was the most successful in enriching for tumor reactive CD4+ T-cells with a median of 37.2%. followed by ADGRG-1 (25.9%), CD57 (19.4%). ICOS (18.2%), PD-1 (16.9%). TIM-3 (13.7%) and CD39 (12.2%). Comparing this to currently published signatures, PD-l / ICOS co-expression was not as successful (18.5%) in enriching NeoCD4+ reactivity7as other single markers. The most successful marker combinations were ADGRG-1 / CD 86 / PD-l and ADGRG-1 / CD86 which had a median enrichment of 44. 12%.

[0191] To gain further insight into cell surface marker profiles, the median fluorescence intensities (MFI) of the experimentally validated anti-tumor, NeoCD4 TCRs were identified. Within the ADGRG-1 population, it was observed that the vast majority of NeoCD4+ T-cells also co-express PD-land ICOS (76.9%) compared to CD86 (20.8%). CD86 co-expression was diminished on screened negative cells (6.0%) compared to PD-l / ICOS (45.4%). Within the CD86 population, PD-l / ICOS co-expression was found on 74.4% of NeoCD4 cells, equivocal to ADGRG-1 co-expression (76.8%). Amongst negatively screened cells, ADGRG-1 co-expression was slightly greater than PD-l / ICOS (54.0% vs. 44.62%). Protein co-expression within the PD-l / ICOS population reveals amongst NeoCD4+ Cells, ADGRG-1 was expressed on 81. 1 % of cells, compared to CD86 (28.8%). Amongst negatively screened cells, CD86 and ADGRG-1 expression was similar (48% vs. 52%). Finally, analyzing CD86 / ADGRG-1 co-expression, it was observed that the majority' of NeoCD4+ cells also coexpressed PD-l / ICOS (80.0%). However, most negatively screened cells also expressed these protein markers (60.0%).EXAMPLE 6

[0192] This example demonstrates the isolation of an anti-Q61R RAS TCR from the T cells from sample 4342.

[0193] NeoCD4+ T-cells were identified for the historic sample 4342, as described in Example 3, including NeoCD4+ T-cells which recognized Q61R RAS. Single cell sequencing was carried out on the Q61R RAS-reactive T cells, as described in the methods section. 4342 TCR 8 was identified (TRAV12-2*01 / TRBV19*01).

[0194] The amino acid sequences of the alpha and beta chain variable regions of the 4342 TCR 8 were obtained, as described in the methods section, and are shown in Table 7. The CDRs are underlined. The N-terminal signal peptides are in bold font.TABLE 7EXAMPLE 7

[0195] This example demonstrates the isolation of an anti-G13D RAS TCR from the T cells from sample 4400.

[0196] NeoCD4+ TCRs were found within sample 4400 (9), as described in Example 4, including four TCRs which recognized G13D RAS (Table 8).TABLE 8

[0197] The amino acid sequences of the alpha and beta chain variable regions for each TCR were obtained, as described in the methods section, and are shown in Table 9. The CDRs are underlined. The N-terminal signal peptides are in bold font.TABLE 9EXAMPLE 8

[0198] This example demonstrates the construction of retroviral vectors encoding the respective TCRs of Examples 6 and 7.

[0199] Full length TRAV and TRBV regions for the 4342 TCR 8 of Table 7 and the 4400 TCR 5, 4400 TCR 1 1, 4400 TCR 37, and 4400 TCR 47 of Table 9 were reconstructed using the IMGT database and a modified murine TRAC and TRBC sequence, as described in the methods section. Nucleotide sequences encoding the a and p chains of each respective TCR were codon-optimized and subcloned into the pMSGVl vector with a 2A peptide between the TCRa and TCR0 chains, as described in the methods section.

[0200] The full length a and (3 chains of each of the five TCRs, including these modifications to the constant region, are shown in Table 10. In Table 10, the CDRs are underlined, and the modified amino acid residues of the constant region are underlined and in bold.TABLE 10EXAMPLE 9

[0201] This example demonstrates the identification of the target mutated peptide recognized by 4342 TCR 8.

[0202] Effector cells were healthy donor PBL transduced with the pMSGV I vector encoding the 4342 TCR 8 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells as target cells pulsed with one of eight different pools of peptides (peptide pools (PP) 1-8). The peptide pools contained different specific mutated peptides expressed by the tumor of patient 4342. Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4-1 BB expression by the effector cells was measured by flow cytometry. The results are shown in Table 11. As show n in Table 11, the 4342 TCR 8 recognized a peptide in PP 1.TABLE 11

[0203] To identify which mutated peptide in PPI was recognized by the 4342 TCR 8, patient-specific dendritic cells (target cells) were independently pulsed with each one of the mutated peptides from PP I and PP2, shown in Table 12. The pulsed target cells were cocultured with the effector cells. Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4- IBB expression by the effector cells was measured by flow cytometry'. The results are shown in Table 12. As shown in Table 12. the 4342 TCR 8 recognized the mutated RAS peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89), which was present in PPLTABLE 12EXAMPLE 10

[0204] This example demonstrates that PBL transduced with the 4342 TCR 8 specifically recognize RAS Q61R.

[0205] Effector cells were healthy donor PBL transduced with the pMSGV 1 vector encoding the 4342 TCR 8 described in Example 8. Effector cells were co-cultured with Patient 4342-specific autologous dendritic cells (target cells) pulsed for 18 hours with 10, 1, 0.1, 0.01. or 0.001 pg / pL of the mutated RAS peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89) or the corresponding WT RAS peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 90) at a 1 : 1 ratio of effector cells to target cells (3E4:3E4). EBV B cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls.

[0206] 4- IBB expression by the effector cells was measured by flow cytometry (Table13). As shown in Table 13, the 4342 TCR 8 specifically recognized the mutated RAS peptide and did not recognize the corresponding WT RAS peptide.TABLE 13EXAMPLE 11

[0207] This example demonstrates that the 4342 TCR 8 recognizes RAS Q61R presented by aHLA-DQAl*0I:01:HLA-DQBl*05:01 heterodimer.

[0208] Effector cells were healthy donor PBL transduced with the pMSGV 1 vector encoding the 4342 TCR 8 described in Example 8. Target cells were COS cells independently transduced with the HLA Class II heterodimers expressed by Patient 4342 shown in Tables 14A-14B. The transduced COS cells were pulsed with the mutated RAS peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89). Effector cells were co-cultured with the peptide-pulsed COS cells at a ratio of 1: 1 (effector cells to target cells). Target cells pulsed with DMSO, and COS cells cultured alone, and effector cells cultured alone (media) served as controls. 4-1BB expression by the effector cells was measured by flow cytometry (Table 14A). IFN-gamma secrection was measured by ELISpot assay (Table 14B). As shown in Table 14A, the 4342 TCR 8 recognized the RAS Q61R peptide presented by aHLA-DQAl*01:01 / HLA-DQBl*05:01 heterodimer.TABLE 14ATABLE 14BEXAMPLE 12

[0209] This example demonstrates the identification of the target mutated peptide recognized by 4400 TCR 5.

[0210] Effector cells were healthy donor PBL transduced with the pMSGV 1 vector encoding the 4400 TCR 5 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with combinations of different pools of peptides, WT KRAS, or mutated KRAS, as shown in Table 15. A cell line derived from implantation of the patient’s tumor into a mouse model also served as target cells (xenograft). The peptide pools contained different specific mutated peptides expressed by the tumor of patient 4400. Because Patient 4400 had previous TCRs that recognized mutated KRAS, WT (MTEYKLVVVGAGGVGKSALTIQLIQ) (SEQ ID NO: 92) and mutated (MTEYKLVVVGAGDVGKSALTIQLIQ) (SEQ ID NO: 91) KRAS were also tested in this experiment. Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4- IBB expression by the effector cells was measured by flow cy tometry7. The results are shown in Table 15. As shown in Table 15, the 4400 TCR 5 recognized mutated KRAS (MTEYKLVVVGAGDVGKSALTIQLIQ) (SEQ ID NO: 91) in PP7.TABLE 15EXAMPLE 13

[0211] This example demonstrates that PBL transduced with the 4400 TCR 5 specifically recognize RAS G13D.

[0212] Effector cells were healthy donor PBL were transduced with the pMSGVl vector encoding the 4400 TCR 5 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with 5e'2, 5e‘3, 5e'4, 5e'5, or 5e‘6pg / pL of the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91) or the corresponding WT RAS peptide of MTEYKLVVVGAGGVGKS ALTIQLIQ (SEQ ID NO: 92). Target cells pulsed with DMSO, effector cells treated with PMA. and effector cells cultured alone (media) served as controls. 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 16. As shown in Table 16, the 4400 TCR 5 specifically recognized the mutated RAS peptide and did not recognize the corresponding WT RAS peptide at concentrations of 5e'3pg / pL and higher.TABLE 16EXAMPLE 14

[0213] This example demonstrates that the 4400 TCR 5 recognizes RAS G13D presented by a HLA-DQA1 *05 :05:HLA-DQB 1*03:01 heterodimer.

[0214] Effector cells were healthy donor PBL were transduced with the pMSGV 1 vector encoding the 4400 TCR 5 described in Example 8. Target cells were COS cells independently transduced with the HLA Class II heterodimers expressed by Patient 4400 shown in Table 17 and pulsed with the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4- IBB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 17. As shown in Table 17. the 4400 TCR 5 recognized the RAS G13D peptide presented by a HLA-DQAl*05:05:HLA-DQBl*03:01 heterodimer.TABLE 17EXAMPLE 15

[0215] This example demonstrates the identification of the target mutated peptide recognized by 4400 TCR 11.

[0216] Effector cells were healthy donor PBL were transduced with the pMSGV 1 vector encoding the 4400 TCR 11 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with combinations of different pools of peptides, WT KRAS, or mutated KRAS, as shown in Table 18. The peptide pools contained different specific mutated peptides expressed by the tumor of patient 4400. Because Patient 4400 had previous TCRs that recognized mutated KRAS. WT (MTEYKLVVVGAGGVGKSALTIQLIQ) (SEQ ID NO: 92) and mutated(MTEYKLVVVGAGDVGKSALTIQLIQ) (SEQ ID NO: 91) KRAS were also tested in this experiment. A cell line derived from implantation of the patient’s tumor into a mouse model also served as target cells (xenograft). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4- IBB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 18. As shown in Table 18, the 4400 TCR 11 recognized mutated KRAS (MTEYKLVVVGAGDVGKSALTIQLIQ) (SEQ ID NO: 91) in PP7.TABLE 18EXAMPLE 16

[0217] This example demonstrates that PBL transduced with the 4400 TCR 11 specifically recognize RAS G13D.

[0218] Effector cells were healthy donor PBL were transduced with the pMSGV 1 vector encoding the 4400 TCR 11 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with 5e'2, 5e'3, 5e'4, 5e'5, or 5e'6pg / gL of the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91) orthe corresponding WT RAS peptide of MTEYKLVVVGAGGVGKSALTIQLIQ (SEQ ID NO: 92). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 19. As shown in Table 19. the 4400 TCR 11 specifically recognized the mutated RAS peptide and did not recognize the corresponding WT RAS peptide.TABLE 19EXAMPLE 17

[0219] This example demonstrates that the 4400 TCR 11 recognizes RAS G13D presented by a HLA-DQAl*05:05:HLA-DQBl*03:01 heterodimer.

[0220] Effector cells were healthy donor PBL were transduced with the pMSGVl vector encoding the 4400 TCR 11 described in Example 8. Target cells were COS cells independently transduced with the HLA Class II heterodimers expressed by Patient 4400 shown in Table 20 and pulsed with the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4- IBB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 20. As shown in Table 20, the 4400 TCR 11 recognized the RAS G13D peptide presented by a HLA-DQAl*05:05:HLA-DQBl*03:01 heterodimer.TABLE 20EXAMPLE 18

[0221] This example demonstrates the identification of the target mutated peptide recognized by 4400 TCR 37.

[0222] Effector cells were healthy donor PBL were transduced with the pMSGVl vector encoding the 4400 TCR 37 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with combinations of different pools of peptides, WT KRAS, or mutated KRAS, as shown in Table 21. The peptide pools contained different specific mutated peptides expressed by the tumor of patient 4400. Because Patient 4400 had previous TCRs that recognized mutated KRAS, WT (MTEYKLVVVGAGGVGKSALTIQLIQ) (SEQ ID NO: 92) and mutated (MTEYKLVVVGAGDVGKSALTIQLIQ) (SEQ ID NO: 91) KRAS were also tested in this experiment. Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. A cell line derived from implantation of the patient’s tumor into a mouse model also served as target cells (xenograft). 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 21 . As shown in Table 21, the 4400 TCR 37 recognized mutated KRAS (MTEYKLVVVGAGDVGKSALTIQLIQ) (SEQ ID NO: 91) in PP7.TABLE 21EXAMPLE 19

[0223] This example demonstrates that PBL transduced with the 4400 TCR 37 specifically recognize RAS G13D.

[0224] Effector cells were healthy donor PBL transduced with the pMSGV 1 vector encoding the 4400 TCR 37 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with 5e'2, 5e'3, 5e'4, 5e'5, or 5e'6pg / pL of the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91) or the corresponding WT RAS peptide of MTEYKLVVVGAGGVGKS ALTIQLIQ (SEQ ID NO: 92). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4-1BB expression by CD4+ effector cells was measured by flow cytometry'. The results are shown in Table 22. As shown in Table 22, the 4400 TCR 37 specifically recognized the mutated RAS peptide and did not recognize the corresponding WT RAS peptide at pulsed peptide concentrations of 5e’3pg / pL or higher.TABLE 22EXAMPLE 20

[0225] This example demonstrates that the 4400 TCR 37 recognizes RAS G13D presented by a HLA-DQAl*05:05:HLA-DQBl*03:01 heterodimer.

[0226] Effector cells w ere healthy donor PBL were transduced with the pMSGV 1 vector encoding the 4400 TCR 37 described in Example 8. Target cells were COS cellsindependently transduced with the HLA Class II heterodimers expressed by Patient 4400 shown in Table 23 and pulsed with the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 23. As shown in Table 23, the 4400 TCR 37 recognized the RAS G13D peptide presented by a HLA-DQAl*05:05:HLA-DQBl*03:01 heterodimer.TABLE 23EXAMPLE 21

[0227] This example demonstrates the identification of the target mutated peptide recognized by 4400 TCR 47.

[0228] Effector cells were healthy donor PBL were transduced with the pMSGV 1 vector encoding the 4400 TCR 47 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with combinations of different pools of peptides, WT KRAS, or mutated KRAS, as shown in Table 24. The peptide pools contained different specific mutated peptides expressed by the tumor of patient 4400. Because Patient 4400 had previous TCRs that recognized mutated KRAS, WT (MTEYKLVVVGAGGVGKSALTIQLIQ) (SEQ ID NO: 92) and mutated MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91) KRAS were also tested in this experiment. Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. A cell line derived from implantation of the patient's tumor into a mouse model also served as target cells (xenograft). 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 24.As shown in Table 24. the 4400 TCR 47 recognized mutated KRAS MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91) in PP7.TABLE 24EXAMPLE 22

[0229] This example demonstrates that PBL transduced with the 4400 TCR 47 specifically recognize RAS G13D.

[0230] Effector cells were healthy donor PBL transduced with the pMSGV 1 vector encoding the 4400 TCR 47 described in Example 8. Effector cells were co-cultured with patient-specific dendritic cells (target cells) pulsed with 5e'2, 5e'3, 5e'4, 5e'5, or 5e'6pg / pL of the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91) or the corresponding WT RAS peptide of MTEYKLVVVGAGGVGKS ALTIQLIQ (SEQ ID NO: 92). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 25. As shown in Table 25, the4400 TCR 47 specifically recognized the mutated RAS peptide and did not recognize the corresponding WT RAS peptide.TABLE 25EXAMPLE 23

[0231] This example demonstrates that the 4400 TCR 47 recognizes RAS G13D presented by a HLA-DQAl*05:01:HLA-DQBl*03:01 heterodimer.

[0232] Effector cells were healthy donor PBL were transduced with the pMSGVl vector encoding the 4400 TCR 47 described in Example 8. Target cells were COS cells independently transduced with the HLA Class II heterodimers expressed by Patient 4400 shown in Table 26 and pulsed with the mutated RAS peptide of MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91). Target cells pulsed with DMSO, effector cells treated with PMA, and effector cells cultured alone (media) served as controls. 4-1BB expression by CD4+ effector cells was measured by flow cytometry. The results are shown in Table 26. As shown in Table 26, the 4400 TCR 47 recognized the RAS G13D peptide presented by a HLA-DQAl*05:01 :HLA-DQBl*03:01 heterodimer.TABLE 26EXAMPLE 24

[0233] This example demonstrates the isolation of an anti-G12D RAS TCR from the TIL of cancer patient 4589.

[0234] T cell exhaustion transcriptomic signature (NeoTCR signature, Lowery et al., Science, 375(6583):877-884 (2022)), was used to predict candidate anti-tumor TCRs from single cell transcriptomic sequencing from 4589 tumor digests. Candidate TCRs were tested against all mutated peptides from the tumor including mutated KRAS candidate peptide, to identify the KRAS G12D-specific TCR (4589 NEO4 TCR 1).

[0235] The amino acid sequences of the alpha and beta chain variable regions are shown in Table 27. The CDRs are underlined. The N-terminal signal peptides are in bold font.TABLE 27EXAMPLE 25

[0236] This example demonstrates the isolation of anti-G12V RAS TCRs from the TIL of cancer patient 4424.

[0237] T cell exhaustion transcriptomic signature (NeoTCR signature, Lowery et al., Science, 375(6583):877-884 (2022)), in combination with DNA-barcoded HLA-multimers constructed using KRAS G12V candidate neopeptides predicted to bind to patient-specific HLA alleles (including A* 11 :01), was used to predict candidate anti-tumor TCRs from singlecell transcriptomic sequencing from 4424 tumor digests. Candidate TCRs were tested against all mutated peptides from the tumor including mutated KRAS candidate peptide, to identify KRAS G12V-specific TCRs.

[0238] The isolated TCRs are shown in Table 28.TABLE 28

[0239] The amino acid sequences of the alpha and beta chain variable regions are shown in Table 29. The CDRs are underlined. The N-terminal signal peptides are in bold font.TABLE 29EXAMPLE 26

[0240] This example demonstrates the construction of retroviral vectors encoding the respective TCRs of Examples 24 and 25.

[0241] Nucleotide sequences encoding the variable regions of the a and P chains of the TCRs of Tables 27 and 29 were obtained and codon-optimized. The TCRp VDJ regions were fused to the mouse TCRP constant chain. The TCRa VJ regions were fused to the mouse TCRa constant chain. Without being bound to a particular theory or mechanism, it is believed that replacing the constant regions of the human TCRa and TCRp chains with the corresponding murine constant regions improves TCR expression and functionality (Cohen et al.. Cancer Res., 66(17): 8878-86 (2006)).

[0242] In addition, the murine TCRa and TCRp constant chains were cysteine-modified. Transmembrane hydrophobic mutations were introduced into the murine TCRa constant chain. Without being bound to a particular theory or mechanism, it is believed that these modifications result in preferential pairing of the introduced TCR chains and enhanced 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)). The full length a and chains of each of the four TCRs. including these modifications to the constant region, are shown in Table 30. In Table 30, the CDRs are underlined, the constant regions are italicized, and the modified amino acid residues of the constant region are underlined and in bold.TABLE 30

[0243] Nucleotide sequences encoding the variable regions of the a and 0 chains of the TCRs of Table 30 were independently cloned into a MSGVl-based retroviral vectors with the following expression cassette configuration: 5’NcoI-VDjp-mC0-Furin / SerGly / P2A-VJa- mCa-EcoRI3’.

[0244] The TCR0 and TCRa chains were separated by a Furin Ser / Gly P2A linker peptide (SEQ ID NO: 93). Without being bound to a particular theory or mechanism, it is believed that the linker peptide provides comparable expression efficiency of the two chains (Szymczak et al.. Nat. Biotechnol.. 22(5):589-94 (2004)).EXAMPLE 27

[0245] This example demonstrates that healthy donor PBL transduced with the retroviral vector encoding the 4589 NEO4 TCR 1 of Example 26 specifically recognize G12D.

[0246] Effector cells were healthy donor PBL transduced with the retroviral vector encoding the 4589 NEO4 TCR 1 of Example 26.

[0247] Target cells were DCs from Patient 4589 pulsed with various concentrations of the G12D peptide of MTEYKLVVVGADGVGKS ALTIQLI (SEQ ID NO: 95) or thecorresponding WT peptide of SEQ ID NO: MTEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 96), as shown in Fig. 1 .

[0248] Target cells were co-cultured with effector cells at an effector to target ratio of 1 : 1. The percentage of CD3+, murine TCR constant region (mTCR) positive cells expressing 4-1BB was measured by flow cytometry. As shown in Fig. 1, the 4589 NEO4 TCR 1 specifically recognized G12D.EXAMPLE 28

[0249] This example demonstrates that cells transduced with the 4424 TCR 12 or the 4424 TCR 13B bind only to VVVGAVGVGK (SEQ ID NO: 99) and not to VVGAVGVGK (SEQ ID NO: 97), and that cells transduced with the 4424 TCR 11, 4424 TCR 17, and 4424 TCR 18 bind to either VVGAVGVGK (SEQ ID NO: 97) or VVVGAVGVGK (SEQ ID NO: 99).

[0250] Healthy donor PBL were independently transduced with a retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 of Example 26.

[0251] The TCR-transduced T cells were stained with fluorescent HLA-A* 11 :01 RAS G12V tetramers containing the predicted 9-mer neoepitope having the amino acid sequence of VVGAVGVGK (SEQ ID NO: 97) or the predicted 10-mer neoepitope having the amino acid sequence of VVVGAVGVGK (SEQ ID NO: 99) in two different colors, provided by the dyes allophycocyanin (APC) and phycoerythrin (PE). The cells were pre-gated on CD8+ mTCR+ (murine TCR constant region, i.e., the TCR-transduced fraction). The percentages of cells stained with either dye were measured by flow cytometry. The results showed that the 4424 TCR 12 and the 4424 TCR 13B bound only to VVVGAVGVGK (SEQ ID NO: 99) and not to VVGAVGVGK (SEQ ID NO: 97). as shown in Table 31 and Fig. 2. The results also showed that the 4424 TCR 11, 4424 TCR 17, and 4424 TCR 18 were able to bind to either VVGAVGVGK (SEQ ID NO: 97) or VVVGAVGVGK (SEQ ID NO: 99), as shown in Table 31 and Fig. 2.TABLE 31

[0252] This HLA-specific tetramer assay shows that 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, and 4424 TCR 18 are HLA-A* 11:01 restricted because the fluorescent HLA tetramers use the HLA-A* 11 :01 molecule.EXAMPLE 29

[0253] This example demonstrates the avidity of the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 with respect to recognizing the G12V 9-mer peptide VVGAVGVGK (SEQ ID NO: 97).

[0254] Healthy donor PBL (effector cells) were independently transduced with the retroviral vector of Example 26 encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18.

[0255] Allogenic, HLA-matched EBV -transformed B cells (target cells) were pulsed for 18 hours with the G12V peptide VVGAVGVGK (SEQ ID NO: 97) or the corresponding WT peptide VVGAGGVGK (SEQ ID NO: 98) at a concentration of 10, 1, 0.1, 0.001, 0.0001, or 0.00001 pg / mL. Target cells treated with phorbol myristate acetate (PMA) served as a positive control. Target cells treated with dimethyl sulfoxide (DMSO) served as a negative control. Cells transduced with the existing clinically utilized 4148 TCR 2 (Cafri et al., Nat. Comm., 10(l):449 (2018)) also served as a control. The target cells were co-cultured with effector cells at an effector to target ratio of 1 : 1. Reactivity was tested by IFNy-secretion using ELISpot assay.

[0256] The ELISpot assay results showed that:• 4424 TCR 11 recognized cells pulsed with 0.001 pg / mL peptide or higher;• 4424 TCR 12 did not recognize any of the target cells;• 4424 TCR 13B recognized cells pulsed with 10 pg / mL peptide;• 4424 TCR 17 and 4148 2 control TCR recognized cells pulsed with0.00001 pg / mL peptide or higher; and• 4424 TCR 18 recognized cells pulsed with 0.0001 pg / mL peptide or higher.

[0257] Reactivity was also tested by measuring the expression of 4- IBB by flow cytometry assay gated on CD8+ / mTCR+ cells (Figures 3A-3E). The results showed that 4424 TCR 11, 4424 TCR 17, and 4424 TCR 18 demonstrated avid recognition of the G12V 9-mer peptide VVGAVGVGK (SEQ ID NO: 97).EXAMPLE 30

[0258] This example demonstrates the avidity of the 4424 TCR 1 1, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 with respect to recognizing the G12V 10-mer peptide VVVGAVGVGK (SEQ ID NO: 99).

[0259] Healthy donor PBL (effector cells) were independently transduced with the retroviral vector of Example 26 encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18.

[0260] Allogenic, HLA-matched EBV -transformed B cells (target cells) were pulsed for 18 hours with the G12V 10-mer peptide VVVGAVGVGK (SEQ ID NO: 99) or the corresponding WT peptide VVVGAGGV GK (SEQ ID NO: 100) at a concentration of 10, 1, 0.1, 0.001, 0.0001, or 0.00001 pg / mL. Target cells treated with PMA served as a positive control. Target cells treated with DMSO served as a negative control. Cells transduced with the 4148 TCR 2 also served as a control. The target cells were co-cultured with effector cells at an effector to target ratio of 1: 1. Reactivity was tested by IFNy-secretion using ELISpot assay.

[0261] The ELISpot assay results showed that the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, 4424 TCR 18 and control 4148 TCR 2 recognized cells pulsed with 0.00001 pg / mL peptide or higher.

[0262] Reactivity was also tested by measuring the expression of 4- IBB by flow cytometry assay gated on CD8+ / mTCR+ cells (Figures 4A-4E). The results showed that4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 demonstrated avid recognition of the G12V 10-mer peptide VVGAVGVGK (SEQ ID NO: 99).EXAMPLE 31

[0263] This example demonstrates that the 4589 NEO4 TCR 1 is restricted by HLA- DRA1 *01 :01 :HLA-DRBl *04:05.

[0264] Effector cells were T cells transduced with the retroviral vector encoding the 4589 NEO4 TCR 1 of Example 26. Target cells were COS cells transfected with the HLA Class II heterodimers indicated in Figure 5 and pulsed with the G12D peptide MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95).

[0265] After co-culture of the target cells with the effector cells, the IFN-gamma secretion was measured by ELISPOT assay. As shown in Figure 5, the 4589 NEO4 TCR 1 was restricted by HLA-DRAl*01:01:HLA-DRBl*04:05.EXAMPLE 32

[0266] This example demonstrates the mutation specificity of 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17. and 4424 TCR 18 by titration assay.

[0267] Healthy donor PBL were sorted for CD4+ or CD8+ expression. Sorted cells were independently transduced with the retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 of Example 26 (effector cells). Sorted PBL transduced with existing clinically -utilized 4148 TCR 2 (Cafri et al., Nat. Comm.. 10(l):449 (2018)) served as control effector cells.

[0268] Allogenic, HLA-matched EBV-transformed B cells (target cells) were pulsed for 18 hours with the 9-mer G12V peptide VVGAVGVGK (SEQ ID NO: 97) or the corresponding WT peptide VVGAGGVGK (SEQ ID NO: 98) at a concentration of 10 pg / mL. 1 pg / mL, 0.1 pg / mL, 0.01 pg / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL or 1 pg / mL. The pulsed target cells were co-cultured with the effector cells. Reactivity was assessed by measuring 4-1BB upregulation by FACS. 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, 4424 TCR 18 and 4148 TCR 2 demonstrated a spectrum of mutation specificity as measured by this titration assay (Figures 6A-6F).EXAMPLE 33

[0269] This example demonstrates the relative dependence or relative independence of 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, and 4424 TCR 18 on the CD8 co-receptor for the recognition of a tumor cell line.

[0270] Healthy donor PBL were sorted for CD3+, CD4+ or CD8+ expression. Sorted cells were independently transduced with the retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B. 4424 TCR 17, or 4424 TCR 18 of Example 26 (effector cells). Sorted PBL transduced with 4148 TCR 2 served as control effector cells.

[0271] A RAS G12V positive, human colorectal cancer tumor line engineered to express HLA A* 11:01 served as target cells.

[0272] The target cells were co-cultured with the effector cells. Reactivity was assessed by measuring 4-1BB upregulation by FACS. 4424 TCR 11, 4424 TCR 13B. , and 4424 TCR 18 were more dependent on the CD8 co-receptor for the recognition of the tumor cell line, while 4424 TCR 12 and 4424 TCR 17 w ere less dependent on the CD8 co-receptor for the recognition of the tumor cell line. 4424 TCR 17 was the most coreceptor independent 9-mer specific TCR among those tested. 4424 TCR 12 was the most coreceptor independent 10- mer specific TCR among those tested. 4424 TCR 12 provided superior recognition of the tumor cell line in CD3+ and CD4+ T cells as compared to the 4148 TCR 2 (Fig. 7).

[0273] In a separate experiment, 4424 TCR 12 and 4424 TCR 17 were shown to be more CD8 co-receptor independent (i.e., have functional performance in both CD8+ and CD4+ T cells) by tetramer and CD8 / CD4 staining (Fig. 13). The 4148 TCR 2 and 4424 TCR13B show ed more CD8 coreceptor dependence (Fig. 13).EXAMPLE 34

[0274] This example demonstrates the relative dependence or relative independence of 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, and 4424 TCR 18 on the CD8 co-receptor for the recognition of a patient-derived organoid.

[0275] Healthy donor PBL were sorted for CD4+ or CD8+ expression. Sorted cells were independently transduced with the retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17. or 4424 TCR 18 of Example 26 (effector cells). Sorted PBL transduced with 4148 TCR 2 served as control effector cells.

[0276] Target cells were naturally occurring, human patient-derived tumor organoids naturally expressing the RAS G12V mutation. Tumor organoids derived from cancer Patient4626 (4626 organoid) (cholangiocarcinoma) or Patient 4424 (4424 organoid) (colorectal cancer) were either pretreated with interferon gamma to upregulate antigen presentation (+IFN-y) or did not receive any pre-treatment. The tumor organoid derived from cancer Patient 4424 served as a negative control (4424 organoid). This negative control tumor organoid lost expression of HL A- A* 11 :01, which abolished regognition by HLA-A* 11 birestricted TCRs. The 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 were obtained from this same patient (Patient 4424) from which this negative control tumor organoid was derived.

[0277] The target cells were co-cultured with the effector cells. Reactivity was assessed by measuring 4-1BB upregulation by FACS. 4424 TCR 11, 4424 TCR 13B. 4424 TCR 17, and 4424 TCR 18 were more dependent on the CD8 co-receptor for the recognition of the tumor orgnoid 4626, while 4424 TCR 12 was less dependent on the CD8 co-receptor for the recognition of the tumor orgnoid 4626 (Figs. 8A-8B). 4424 TCR 12 provided superior recognition of the 4626 organoid by CD8+ and CD4+ T cells as compared to the 4148 TCR 2 (Figs. 8A-8B).EXAMPLE 35

[0278] This example demonstrates that cells transduced with the 4424 TCR 12 delay tumor progression in vivo.

[0279] On Day 0, NOD scid gamma mice were subcutaneously flank-injected with KRAS G12V (A* 11 :01) colorectal cancer patient-derived xenograft cells (4391 PDX + HLA- A11). On Day 0, healthy donor PBL were independently transduced with the retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 of Example 26 (effector cells). PBL transduced with 4148 TCR 2 served as control effector cells. Transduced cells were enriched for CD8 expression by depleting the population of CD4+ cells. CD8+ transduced cells were nonspecifically stimulated with anti- CD3 antibody. On Day 14, the number of transduced cells was expanded using 300 lU / mL IL-2. On Day 14, tumor-bearing mice were injected (via tail vein) with the expanded number (1E7) of transduced cells (5 mice per group). Tumor size was measured on Days 0-91. A schematic illustrating the methods used in the experiment described in this Example is shown in Fig. 9.

[0280] Cells transduced with the 4424 TCR 12 delayed tumor progression in vivo (Fig. 10). 4424 TCR 12 showed superior in vivo tumor regression against 4391 PDX compared to 4148 TCR 2.EXAMPLE 36

[0281] This example demonstrates that 4424 TCR 12 shows superior recognition of the 8045 organoid by CD8+ and CD4+ T cells as compared to the the 4148 TCR 2.

[0282] Healthy donor PBL were sorted for CD4+ or CD8+ expression. Sorted cells were independently transduced with the retroviral vector encoding the 4424 TCR 11, 4424 TCR 12, 4424 TCR 13B, 4424 TCR 17, or 4424 TCR 18 of Example 26 (effector cells). Sorted PBL transduced with 4148 TCR 2 serv ed as control effector cells.

[0283] Target cells were axenograft derived from colorectal cancer patient 8045 (PDX) or tumor organoids derived from Patient 8045 (8045 organoid) or colorectal cancer Patient 4447 (4447 organoid) naturally expressing the RAS G12V mutation. The organoids were either pretreated with interferon gamma to upregulate antigen presentation (+IFN-y) or did not receive any pre-treatment. The xenograft was not pretreated. The tumor organoid derived from cancer Patient 4447 had an irrelevant HLA-mutation combination and served as a negative control.

[0284] The target cells were co-cultured with the effector cells. Reactivity was assessed by measuring 4-1BB upregulation by FACS. 4424 TCR 12 showed superior recognition of the 8045 organoid by CD8+ and CD4+ T cells as compared to the the 4148 TCR 2 (Figs. 11A-11B).EXAMPLE 37

[0285] This example demonstrates that PBL transduced with the 4342 TCR 8 specifically recognize the RAS Q61R neoepitope and is restricted by HL A DQA1 *01:02- DQBl*05:02 / DQBl*02:02 or DQAl*01:01-DQBl*05:01 / DQBl*03:01.

[0286] The tumor of Patient 4342 expressed the NRAS Q61R mutation. The following experiment was performed to determine whether the 4342 TCR 8 can specifically recognize the RAS Q61R neoepitope.

[0287] Target cells were APCs from Patients 4508 and 4487. The tumor of Patient 4508 expressed the KRAS Q61R mutation. Patient 4508 had the HLA-genotype of DQAl*01 :02- DQB1*O5:O2 / DQB1 *02:02. The tumor of Patient 4487 did not express the KRAS Q61Rmutation. Patient 4487 had the HLA-genotype of DQA1*O1:O1-DQB1*O5:O1 / DQB1*O3:O1, which was the same genotype as the patient from which the 4342 TCR 8 was isolated. The target cells were pulsed for 18h with a pool of 25-mer peptides containing the RAS Q61R neoepitope ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89).

[0288] Effector cells were healthy donor PBL were transduced with the pMSGV 1 vector encoding the 4342 TCR 8 described in Example 8. Effector cells were independently cocultured with the pulsed target cells from Patient 4508 or Patient 4487 at a ratio of 1:1 (target cells to effector cells) (3E4:3E4). DMSO pulsed EBV B cells were used as control targets while PMA was used as a positive control. 4- IBB activation on TCR-transduced T cells (gated on CD3+ T cells (APC-C77)) was measured by flow cytometry.

[0289] The results showed that 4342 TCR 8 recognized RAS Q61R neoepitopes containing the same 25-mer sequence ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89) and is restricted by HLA DQAl*01:02-DQBl*05:02 / DQBl*02:02 or DQA1*O1 :O1-DQB1*O5:O1 / DQB1*O3:O1 (Table 32).TABLE 32EXAMPLE 38

[0290] This example demonstrates that the 4342 TCR 8 specifically recognizes the RAS Q61R neoepitope and is restricted by HLA DQAl *01 :02-DQB1 *O5:O2 / DQB1 *02:02 or DQA1*O1:O1-DQB1*O5:O1 / DQB1*O3:O1.

[0291] 4342 TCR 8 was tested for mutation specificity against the RAS Q61R neoepitope. Target cells were APCs from Patient 4508 (DQAl*01 :02- DQBl*05:02 / DQBl *02:02) or Patient 4487 (DQA1*O1 :O1-DQB1*O5:O1 / DQB1*O3:O1). The target cells were pulsed for 18h with 25-mer peptideETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89) containing the RAS Q61R neoepitope or the corresponding WT peptide at one of the following concentrations: 10 pg / mL, 1 pg / mL, 0.1 pg / rnL, lO ng / mL, 1 ng / mL, 0.1 ng / mL. 10 pg / mL, or 1 pg / mL.

[0292] Effector cells were healthy donor PBL transduced with the pMSGVl vector encoding the 4342 TCR 8 described in Example 8. Effector cells were independently cocultured with the pulsed target cells at a ratio of 1 : 1 (target cells to effector cells) (3E4:3E4). The percentage of cells expressing 4-1BB was measured by flow cytometry (Figure 12). The results demonstrated that the 4342 TCR 8 specifically recognized the RAS Q61R neoepitope and is restricted by HLA DQAl*01:02-DQBl*05:02 / DQBl*02:02 or DQA1 *01:01- DQBl*05:01 / DQBl*03:01.EXAMPLE 39

[0293] This example demonstrates that the 4342 TCR 8 specifically recognizes RAS Q61R neoepitope and is restricted by HLA DQA1*O1:O2-DQB1*O5:O2 / DQB1*O2:O2 or DQA1*O1:O1-DQB1*O5:O1 / DQB1*O3:O1.

[0294] The 4342 TCR 8 was tested for HLA-crossreactivity against possible HLA class II combinations. Target cells were APCs from Patient 4508 (DQA1 *01:02- DQBl*05:02 / DQBl*02:02) or Patient 4487 (DQA1*O1 :O1-DQB1*O5:O1 / DQB1*O3:O1). Irrelevant APCs from Patient 4454 (DQAl*01:03-DQBl*03:02 / DQBl*06:03) served as negative control target cells. The target cells were pulsed for 18h with 25-mer peptide ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89) containing the RAS Q61R neoepitope.

[0295] Effector cells were healthy donor PBL transduced with the pMSGVl vector encoding the 4342 TCR 8 described in Example 8. Effector cells were independently cocultured with the pulsed target cells at a ratio of 1 : 1 (target cells to effector cells) (3E4:3E4).

[0296] The percentage of cells expressing 4- IBB was measured by flow cytometry gated on CD3+ T cells (APC-Cy7)). (Table 33).TABLE 33

[0297] The results demonstrated that the 4342 TCR 8 specifically recognized RAS Q61R neoepitope and is restricted by HLA DQAl*01 :02-DQBl*05:02 / DQBl*02:02 orDQA1*O1:O1-DQB1*O5:O1 / DQB1*O3:OLEXAMPLE 40

[0298] This example demonstrates that 4342 TCR 8 specifically recognized RAS Q61R neoepitope and is restricted by HLA DQAl*01 :02-DQBl*05:02 / DQBl*02:02 or DQAl*01:01-DQBl*05:01 / DQBl*03:01.

[0299] The 4342 TCR 8 was tested for HLA-crossreactivity against possible HLA class II combinations. Target cells were APCs from Patient 4508 (DQA1 *01:02- DQBl*05:02 / DQBl*02:02) or Patient 4487 (DQAl*01:01-DQBl*05:01 / DQBl*03:01). Irrelevant APCs from Patient 4454 (DQAl*01:03-DQBl*03:02 / DQBl*06:03) served as negative control target cells. Further negative control target cells included those listed in Table 34. The target cells were pulsed for 18h with 25-mer peptide ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89) containing the RAS Q61R neoepitope.

[0300] Effector cells were healthy donor PBL transduced with the pMSGVl vector encoding the 4342 TCR 8 described in Example 8. Effector cells were independently cocultured with the pulsed target cells at a ratio of 1 : 1 (target cells to effector cells) (3E4:3E4). PMA-treated effector cells served as positive control. The percentage of cells expressing 4- 1BB was measured by flow cytometry (Table 34).TABLE 34

[0301] The results demonstrated that 4342 TCR 8 specifically recognized RAS Q61R neoepitope and is restricted by HLA DQAl*01:02-DQBl*05:02 / DQBl*02:02 or DQAl*01:01-DQBl*05:01 / DQBl*03:0L

[0302] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0303] The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherw ise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, 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 indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate 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.

[0304] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to 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 otherwise clearly contradicted by context.

Claims

1. CLAIM(S):

1. An isolated or purified T-cell receptor (TCR) having antigenic specificity for a mutated human RAS amino acid sequence with a substitution of (i) glycine at position 12 with aspartic acid, (ii) glycine at position 12 with valine, (iii) glycine at position 13 with aspartic acid or (iv) glutamine at position 61 with arginine, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS). or a mutated human Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) ammo acid sequence, wherein positions 12, 13 and 61 are defined by reference to the wild-type human KRAS, wild-type human HRAS, or wild-ty pe human NRAS protein, respectively, and wherein the TCR comprises the amino acid sequences of:(a) all ofSEQ IDNOs: 1-3,(b) all ofSEQ IDNOs: 4-6,(c) all ofSEQ IDNOs: 1-6,(d)all ofSEQ IDNOs: 11-13,(e) all ofSEQ ID NOs: 14-16,(f) all ofSEQ IDNOs: 11-16,(g) all ofSEQ IDNOs: 21-23,(h) all ofSEQ IDNOs: 24-26,(i) all ofSEQ ID NOs: 21-26,(j) all ofSEQ IDNOs: 31-33,(k) all ofSEQ IDNOs: 34-36,(l) all ofSEQ IDNOs: 31-36,(m) all of SEQ ID NOs: 41-43.(n) all ofSEQ ID NOs: 44-46,(o) all ofSEQ IDNOs: 41-46,(p) all ofSEQ IDNOs: 101-103,(q) all ofSEQ IDNOs: 104-106,(r) all ofSEQ IDNOs: 101-106,(s) all ofSEQ IDNOs: 111-113,(t)all ofSEQ IDNOs: 114-116,I l l(u) all of SEQ ID NOs: 111-116.(v) all of SEQ ID NOs: 121-123,(w) all of SEQ ID NOs: 124-126,(x) all of SEQ ID NOs: 121-126,(y) all of SEQ ID NOs: 131-133.(z) all of SEQ ID NOs: 134-136,(aa) all of SEQ ID NOs: 131-136,(bb) all of SEQ ID NOs: 141-143,(cc) all of SEQ ID NOs: 144-146,(dd) all of SEQ ID NOs: 141-146,(ee) all of SEQ ID NOs: 151-153,(ff) all of SEQ ID NOs: 154-156, or(gg) all of SEQ ID NOs: 151-156.

2. The TCR according to claim 1, wherein the mutated human RAS amino acid sequence is ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89), MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91), MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95), VVGAVGVGK (SEQ ID NO:97), or VVVGAVGVGK (SEQ ID NO: 99).

3. The TCR according to claim 1 or 2, wherein the TCR does not have antigenic specificity for the wild-type human RAS amino acid sequence of ETCLLDILDTAGQEEYSAMRDQYMR (SEQ ID NO: 90), MTEYKLVVVGAGGVGKSALTIQLIQ (SEQ ID NO: 92), MTEYKLVVVGAGGVGKSALTIQLI (SEQ ID NO: 96), VVGAGGVGK (SEQ ID NO:98), or VVVGAGGVGK (SEQ ID NO: 100).

4. The TCR according to any one of claims 1-3, wherein the mutated human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) Class II molecule.

5. The TCR according to claim 4. wherein the HLA Class II molecule is an HLA-DQ heterodimer or an HLA-DR heterodimer.

6. The TCR according to claim 4, wherein the HLA Class II molecule is an HLA-DQALHLA-DQBl heterodimer or an HLA-DRA1:HLA-DRB1 heterodimer.

7. The TCR according to claim 4. wherein the HLA Class II molecule is an HLA-DQA1 *01:01 :HLA-DQB 1 *05 :01 heterodimer, an HLA-DQA 1 *05 : 0 :HLA- DQBl*03:01 heterodimer, an HLA-DQAl*05:01 :HLA-DQBl*03:01 heterodimer, an HLA- DRA1 *01 :01:HLA-DRBl *04:05 heterodimer, an HLA-DQAl*01 :01 :HLA-DQBl*03:01 heterodimer, an HLA-DQAl*01:02:HLA-DQBl*05:02 heterodimer, or an HLA-DQA 1*01: 02:HLA-DQB 1 *02: 02 heterodimer.

8. The TCR according to any one of claims 1-3, wherein the mutated human RAS amino acid sequence is presented by a human leukocyte antigen (HLA) Class I molecule.

9. The TCR according to claim 8, wherein the HLA Class I molecule is an HLA- A molecule.

10. The TCR according to claim 8. wherein the HLA Class I molecule is an HLA- Al l molecule.

11. The TCR according to claim 8. wherein the HLA Class I molecule is encoded by the HLA-A*11:01 allele.

12. The TCR according to any one of claims 1-11, comprising the amino acid sequence(s) of:(1) SEQ ID NO: 7,(2) SEQ ID NO: 8,(3) SEQ ID NO: 9,(4) SEQ ID NO: 10,(5) SEQ ID NO: 17,(6) SEQ ID NO: 18,(7) SEQ ID NO: 19,(8) SEQ ID NO: 20,(9) SEQ ID NO: 27,(10) SEQ ID NO: 28,(11) SEQ ID NO: 29,(12) SEQ ID NO: 30,(13) SEQ ID NO: 37,(14) SEQ ID NO: 38,(15) SEQ ID NO: 39,(16) SEQ ID NO: 40,(17) SEQ ID NO: 47,(18) SEQ ID NO: 48,(19) SEQ ID NO: 49,(20) SEQ ID NO: 50,(21) SEQ ID NO: 107,(22) SEQ ID NO: 108,(23) SEQ ID NO: 109,(24) SEQ ID NO: 110,(25) SEQ ID NO: 117,(26) SEQ ID NO: 118.(27) SEQ ID NO: 119,(28) SEQ ID NO: 120,(29) SEQ ID NO: 127,(30) SEQ ID NO: 128.(31 ) SEQ ID NO: 129,(32) SEQ ID NO: 130,(33) SEQ ID NO: 137,(34) SEQ ID NO: 138,(35) SEQ ID NO: 139,(36) SEQ ID NO: 140,(37) SEQ ID NO: 147,(38) SEQ ID NO: 148,(39) SEQ ID NO: 149.(40) SEQ ID NO: 150,(41) SEQ ID NO: 157.(42) SEQ ID NO: 158,(43) SEQ ID NO: 159,(44) SEQ ID NO: 160,(45) both of SEQ ID NOs: 7 and 8,(46) both of SEQ ID NOs: 9 and 10,(47) both of SEQ ID NOs: 17 and 18,(48) both of SEQ ID NOs: 19 and 20,(49) both of SEQ ID NOs: 27 and 28,(50) both of SEQ ID NOs: 29 and 30,(51) both of SEQ ID NOs: 37 and 38,(52) both of SEQ ID NOs: 39 and 40,(53) both of SEQ ID NOs: 47 and 48,(54) both of SEQ ID NOs: 49 and 50,(55) both of SEQ ID NOs: 107 and 108,(56) both of SEQ ID NOs: 109 and 110,(57) both of SEQ ID NOs: 117 and 118,(58) both of SEQ ID NOs: 119 and 120,(59) both of SEQ ID NOs: 127 and 128.(60) both of SEQ ID NOs: 129 and 130,(61) both of SEQ ID NOs: 137 and 138,(62) both of SEQ ID NOs: 139 and 140,(63) both of SEQ ID NOs: 147 and 148,(64) both of SEQ ID NOs: 149 and 150,(65) both of SEQ ID NOs: 157 and 158, or(66) both of SEQ ID NOs: 159 and 160.

13. The TCR of any one of claims 1-12, further comprising:(a) the amino acid sequence of SEQ ID NO: 63, wherein:(i) X at position 48 of SEQ ID NO: 63 is Thr or Cys;(ii) X at position 112 of SEQ ID NO: 63 is Ser, Ala, Vai, Leu. He. Pro, Phe. Met, or Trp;(iii) X at position 114 of SEQ ID NO: 63 is Met. Ala, Vai. Leu, He, Pro, Phe, or Trp; and(iv) X at position 115 of SEQ ID NO: 63 is Gly, Ala, Vai, Leu, He, Pro, Phe, Met, or Trp;(b) the amino acid sequence of SEQ ID NO: 64, wherein X at position 57 of SEQ ID NO: 64 is Ser or Cys; or(c) both (a) and (b).

14. The isolated or purified TCR of any one of claims 1-13, comprising the amino acid sequence(s) of:(1) SEQ ID NO: 69,(2) SEQ ID NO: 70,(3) SEQ ID NO: 71,(4) SEQ ID NO: 72,(5) SEQ ID NO: 73,(6) SEQ ID NO: 74,(7) SEQ ID NO: 75,(8) SEQ ID NO: 76,(9) SEQ ID NO: 77,(10) SEQ ID NO: 78,(11) SEQ ID NO: 79,(12) SEQ ID NO: 80,(13) SEQ ID NO: 81,(14) SEQ ID NO: 82,(15) SEQ ID NO: 83,(16) SEQ ID NO: 84,(17) SEQ ID NO: 85,(18) SEQ ID NO: 86,(19) SEQ ID NO: 87,(20) SEQ ID NO: 88,(21) SEQ ID NO: 161,(22) SEQ ID NO: 162.(23) SEQ ID NO: 163,(24) SEQIDNO: 164.(25) SEQIDNO: 165,(26) SEQIDNO: 166,(27) SEQIDNO: 167,(28) SEQIDNO: 168,(29) SEQ ID NO: 169,(30) SEQIDNO: 170,(31) SEQIDNO: 171,(32) SEQIDNO: 172,(33) SEQIDNO: 173.(34) SEQIDNO: 174,(35) SEQIDNO: 175,(36) SEQIDNO: 176,(37) SEQIDNO: 177,(38) SEQIDNO: 178,(39) SEQIDNO: 179,(40) SEQIDNO: 180,(41) SEQIDNO: 181,(42) SEQIDNO: 182.(43) SEQIDNO: 183,(44) SEQIDNO: 184,(45) both of SEQ ID NOs: 69 and 70,(46) both of SEQ ID NOs: 71 and 72,(47) both of SEQ ID NOs: 73 and 74,(48) both of SEQ ID NOs: 75 and 76,(49) both of SEQ ID NOs: 77 and 78,(50) both of SEQ ID NOs: 79 and 80,(51) both of SEQ ID NOs: 81 and 82,(52) both of SEQ ID NOs: 83 and 84,(53) both of SEQ ID NOs: 85 and 86,(54) both of SEQ ID NOs: 87 and 88,(55) both of SEQ ID NOs: 161 and 162.(56) both of SEQ ID NOs: 163 and 164,(57) both of SEQ ID NOs: 165 and 166,(58) both of SEQ ID NOs: 167 and 1 8,(59) both of SEQ ID NOs: 169 and 170,(60) both of SEQ ID NOs: 171 and 172,(61) both of SEQ ID NOs: 173 and 174,(62) both of SEQ ID NOs: 175 and 176,(63) both of SEQ ID NOs: 177 and 178,(64) both of SEQ ID NOs: 179 and 180,(65) both of SEQ ID NOs: 181 and 182, or(66) both of SEQ ID NOs: 183 and 184.

15. An isolated or purified polypeptide comprising a functional portion of the TCR of any one of claims 1-14, wherein the functional portion comprises the amino acid sequences of:(a) all of SEQ ID NOs: 1-3,(b) all of SEQ ID NOs: 4-6,(c) all of SEQ ID NOs: 1-6,(d) all of SEQ ID NOs: 11-13,(e) all of SEQ ID NOs: 14-16,(f) all of SEQ ID NOs: 11-16,(g) all of SEQ ID NOs: 21-23,(h) all of SEQ ID NOs: 24-26,(i) all of SEQ ID NOs: 21-26,(j) all of SEQ ID NOs: 31-33,(k) all of SEQ ID NOs: 34-36,(l) all of SEQ ID NOs: 31-36,(m) all of SEQ ID NOs: 41-43.(n) all of SEQ ID NOs: 44-46,(o) all of SEQ ID NOs: 41-46,(p) all of SEQ ID NOs: 101-103,(q) all of SEQ ID NOs: 104-106.(r) all of SEQ ID NOs: 101-106.(s) all of SEQ ID NOs: 1 11-113,(t) all ofSEQ IDNOs: 114-116,(u)all ofSEQ IDNOs: 111-116,(v) all ofSEQ IDNOs: 121-123,(w) all ofSEQ ID NOs: 124-126,(x) all ofSEQ IDNOs: 121-126.(y) all ofSEQ ID NOs: 131-133,(z)all ofSEQ ID NOs: 134-136,(aa) all ofSEQ ID NOs: 131-136,(bb) all of SEQ ID NOs: 141-143,(cc) all of SEQ ID NOs: 144-146,(dd) all of SEQ ID NOs: 141-146,(ee)all of SEQ ID NOs: 151-153,(ff) all ofSEQ ID NOs: 154-156, or(gg)all of SEQ ID NOs: 151-156.

16. The isolated or purified polypeptide according to claim 15, wherein the functional portion comprises the amino acid sequence(s) of:(l) SEQ ID NO: 7,(2) SEQ ID NO: 8.(3) SEQ ID NO: 9,(4) SEQ ID NO: 10,(5) SEQ ID NO: 17,(6) SEQ ID NO: 18,(7) SEQ ID NO: 19,(8) SEQ ID NO: 20,(9) SEQ ID NO: 27,(10) SEQ ID NO: 28,(11) SEQ ID NO: 29,(12) SEQ ID NO: 30,(13) SEQ ID NO: 37,(14) SEQ ID NO: 38,(15) SEQ ID NO: 39,(16) SEQ ID NO: 40,(17) SEQ ID NO: 47,(18) SEQ ID NO: 48,(19) SEQ ID NO: 49,(20) SEQ ID NO: 50,(21) SEQ ID NO: 107,(22) SEQ ID NO: 108,(23) SEQ ID NO: 109,(24) SEQ ID NO: 110,(25) SEQ ID NO: 117,(26) SEQ ID NO: 118.(27) SEQ ID NO: 119,(28) SEQ ID NO: 120,(29) SEQ ID NO: 127,(30) SEQ ID NO: 128,(31) SEQ ID NO: 129,(32) SEQ ID NO: 130,(33) SEQ ID NO: 137,(34) SEQ ID NO: 138,(35) SEQ ID NO: 139.(36) SEQ ID NO: 140,(37) SEQ ID NO: 147,(38) SEQ ID NO: 148,(39) SEQ ID NO: 149.(40) SEQ ID NO: 150,(41) SEQ ID NO: 157,(42) SEQ ID NO: 158,(43) SEQ ID NO: 159,(44) SEQ ID NO: 160,(45) both of SEQ ID NOs: 7 and 8,(46) both of SEQ ID NOs: 9 and 10,(47) both of SEQ ID NOs: 17 and 18,(48) both of SEQ ID NOs: 19 and 20,(49) both of SEQ ID NOs: 27 and 28,(50) both of SEQ ID NOs: 29 and 30,(51 ) both of SEQ ID NOs: 37 and 38,(52) both of SEQ ID NOs: 39 and 40,(53) both of SEQ ID NOs: 47 and 48,(54) both of SEQ ID NOs: 49 and 50,(55) both of SEQ ID NOs: 107 and 108,(56) both of SEQ ID NOs: 109 and 110,(57) both of SEQ ID NOs: 117 and 118,(58) both of SEQ ID NOs: 119 and 120,(59) both of SEQ ID NOs: 127 and 128.(60) both of SEQ ID NOs: 129 and 130,(61) both of SEQ ID NOs: 137 and 138,(62) both of SEQ ID NOs: 139 and 140,(63) both of SEQ ID NOs: 147 and 148,(64) both of SEQ ID NOs: 149 and 150,(65) both of SEQ ID NOs: 157 and 158, or(66) both of SEQ ID NOs: 159 and 160.

17. The isolated or purified polypeptide of claim 15 or 16. further comprising:(a) the amino acid sequence of SEQ ID NO: 63, wherein:(i) X at position 48 of SEQ ID NO: 63 is Thr or Cys;(ii) X at position 112 of SEQ ID NO: 63 is Ser, Ala, Vai, Leu. He. Pro, Phe. Met, or Trp;(iii) X at position 1 14 of SEQ ID NO: 63 is Met, Ala, Vai, Leu, He, Pro, Phe, or Trp; and(iv) X at position 115 of SEQ ID NO: 63 is Gly, Ala, Vai, Leu, He, Pro, Phe, Met, or Trp;(b) the amino acid sequence of SEQ ID NO: 64, wherein X at position 57 of SEQ ID NO: 64 is Ser or Cys; or(c) both (a) and (b).

18. The isolated or purified polypeptide of any one of claims 15-17. comprising the amino acid sequence(s) of:(1) SEQ ID NO: 69,(2) SEQ ID NO: 70,(3) SEQ ID NO: 71,(4) SEQ ID NO: 72,(5) SEQ ID NO: 73,(6) SEQ ID NO: 74,(7) SEQ ID NO: 75,(8) SEQ ID NO: 76,(9) SEQ ID NO: 77,(10) SEQ ID NO: 78,(11) SEQ ID NO: 79,(12) SEQ ID NO: 80,(13) SEQ ID NO: 81,(14) SEQ ID NO: 82,(15) SEQ ID NO: 83,(16) SEQ ID NO: 84,(17) SEQ ID NO: 85,(18) SEQ ID NO: 86,(19) SEQ ID NO: 87,(20) SEQ ID NO: 88,(21) SEQ ID NO: 161,(22) SEQ ID NO: 162,(23) SEQ ID NO: 163.(24) SEQ ID NO: 164,(25) SEQ ID NO: 165,(26) SEQ ID NO: 166,(27) SEQ ID NO: 167,(28) SEQ ID NO: 168,(29) SEQ ID NO: 169,(30) SEQ ID NO: 170,(31) SEQ ID NO: 171,(32) SEQ ID NO: 172.(33) SEQ ID NO: 173,(34) SEQIDNO: 174.(35) SEQIDNO: 175,(36) SEQIDNO: 176,(37) SEQIDNO: 177,(38) SEQIDNO: 178,(39) SEQ ID NO: 179,(40) SEQIDNO: 180,(41) SEQIDNO: 181,(42) SEQIDNO: 182,(43) SEQIDNO: 183.(44) SEQIDNO: 184,(45) both of SEQ ID NOs: 69 and 70,(46) both of SEQ ID NOs: 71 and 72,(47) both of SEQ ID NOs: 73 and 74,(48) both of SEQ ID NOs: 75 and 76,(49) both of SEQ ID NOs: 77 and 78,(50) both of SEQ ID NOs: 79 and 80,(51) both of SEQ ID NOs: 81 and 82,(52) both of SEQ ID NOs: 83 and 84,(53) both of SEQ ID NOs: 85 and 86,(54) both of SEQ ID NOs: 87 and 88,(55) both of SEQ ID NOs: 161 and 162,(56) both of SEQ ID NOs: 163 and 164,(57) both of SEQ ID NOs: 165 and 166,(58) both of SEQ ID NOs: 167 and 168,(59) both of SEQ ID NOs: 169 and 170,(60) both of SEQ ID NOs: 171 and 172,(61) both of SEQ ID NOs: 173 and 174,(62) both of SEQ ID NOs: 175 and 176,(63) both of SEQ ID NOs: 177 and 178,(64) both of SEQ ID NOs: 179 and 180,(65) both of SEQ ID NOs: 181 and 182. or(66) both of SEQ ID NOs: 183 and 184.

19. An isolated or purified protein, comprising:(a) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 1- 3 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 4-6;(b) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs:I I-13 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 14-16;(c) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 21-23 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 24-26;(d) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 31-33 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 34-36;(e) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 41-43 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 44-46;(!) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 101-103 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 104-106;(g) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs:I I I-113 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 114-116;(h) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 121 -123 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 124-126;(i) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 131-133 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 134-136;(j) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 141-143 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 144-146; or(k) a first polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 151 -153 and a second polypeptide chain comprising the amino acid sequences of SEQ ID NOs: 154-156.

20. The isolated or purified protein according to claim 19. wherein:(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: 9 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10;(iii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18;(iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 19 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 20;(v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 27 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 28;(vi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 29 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 30;(vii) 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;(viii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 39 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 40;(ix) 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;(x) 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;(xi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 107 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 108;(xii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 109 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 110;(xiii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 17 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 118;(xiv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 119 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 120;(xv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 127 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 128;(xvi) 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: 130;(xvii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 137 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 138;(xviii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 139 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 140;(xix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 147 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 148;(xx) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 149 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 150;(xxi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 157 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 158; or(xxii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 159 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 160.

21. The isolated or purified protein of claim 19 or 20, wherein:(a) the first polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 63, wherein:(i) X at position 48 of SEQ ID NO: 63 is Thr or Cys;(ii) X at position 112 of SEQ ID NO: 63 is Ser, Ala, Vai, Leu, He, Pro, Phe, Met, or Trp;(iii) X at position 1 14 of SEQ ID NO: 63 is Met, Ala, Vai, Leu, He, Pro, Phe, or Trp; and(iv) X at position 115 of SEQ ID NO: 63 is Gly, Ala, Vai, Leu, He, Pro, Phe, Met, or Trp;(b) the second polypeptide chain further comprises the amino acid sequence of SEQ ID NO: 64, wherein X at position 57 of SEQ ID NO: 64 is Ser or Cys; or(c) both (a) and (b).

22. The isolated or purified protein of any one of claims 19-21, wherein:(i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 70;(ii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 72;(iii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74;(iv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76;(v) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 77 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 78;(vi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 79 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 80;(vii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 81 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82;(viii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:83 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 84;(ix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86;(x) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88;(xi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 161 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 162;(xii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 163 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 164;(xiii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 165 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 166;(xiv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 167 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 168;(xv) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 169 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 170;(xvi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 171 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 172;(xvii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 173 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 174;(xviii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 175 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 176;(xix) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 177 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 178;(xx) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 179 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:(xxi) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 181 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 182; or(xxii) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 183 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 184.

23. A bispecific engager TCR fusion protein comprising (i) the TCR according to any one of claims 1-14, the polypeptide according to any one of claims 15-18, or the protein according to any one of claims 19-23 and (li) an anti-CD3 effector.

24. An isolated or purified nucleic acid comprising a nucleotide sequence encoding the TCR according to any one of claims 1-14, the polypeptide according to any one of claims 15-18, or the protein according to any one of claims 19-23.

25. 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 sequence, respectively, encode the amino sequences of SEQ ID NOs: 7 and 8; 8 and 7; 9 and 10; 10 and 9; 17 and 18; 18 and 17; 19 and 20; 20 and 19; 27 and 28; 28 and 27; 29 and 30; 30 and 29; 37 and 38; 38 and 37; 39 and 40; 40 and 39; 47 and 48; 48 and 47; 49 and 50; 50 and 49; 69 and 70; 70 and 69; 71 and 72; 72 and 71; 73 and 74; 74 and 73; 75 and 76; 76 and 75; 77 and 78; 78 and 77; 79 and 80; 80 and 79; 81 and 82; 82 and 81; 83 and 84; 84 and 83; 85 and 86; 86 and 85; 87 and 88; 88 and 87; 107 and 108; 108 and 107; 109 and 110; 110 and 109; 117 and 1 18; 118 and 117; 119 and 120; 120 and 119; 127 and 128; 128 and 127; 129 and 130; 130 and 129; 137 and 138; 138 and 137; 139 and 140; 140 and 139; 147 and 148;148 and 147; 149 and 150; 150 and 149; 157 and 158; 158 and 157; 159 and 160; 160 and 159; 161 and 162; 162 and 161; 163 and 164; 164 and 163; 165 and 166; 166 and 165; 167 and 168; 168 and 167; 169 and 170; 170 and 169; 171 and 172; 172 and 171; 173 and 174;174 and 173; 175 and 176; 176 and 175; 177 and 178; 178 and 177; 179 and 180; 180 and179; 181 and 182; 182 and 181; 183 and 184; or 184 and 183.

26. The isolated or purified nucleic acid according to claim 25, further comprising a third nucleotide sequence interposed between the first and second nucleotide sequence, wherein the third nucleotide sequence encodes a cleavable linker peptide.

27. The isolated or purified nucleic acid according to claim 26, wherein the cleavable linker peptide comprises the amino acid sequence of RAKRSGSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 93).

28. A recombinant expression vector comprising the nucleic acid according to any one of claims 24-27.

29. The recombinant expression vector according to claim 28, which is a transposon, a retroviral vector, or a lentiviral vector.

30. An isolated or purified TCR, polypeptide, or protein encoded by the nucleic acid according to any one of claims 24-27 or the vector according to claim 28 or 29.

31. An isolated or purified TCR, polypeptide, or protein that results from expression of the nucleic acid according to any one of claims 24-27or the vector according to claim 28 or 29 in a cell.

32. An in vitro method of producing a host cell expressing a TCR that has antigenic specificity for the peptide of ETCLLDILDTAGREEYSAMRDQYMR (SEQ ID NO: 89), MTEYKLVVVGAGDVGKSALTIQLIQ (SEQ ID NO: 91 ), MTEYKLVVVGADGVGKSALTIQLI (SEQ ID NO: 95), VVGAVGVGK (SEQ ID NO: 97), or VVVGAVGVGK (SEQ ID NO: 99). the method comprising contacting a cell with the vector according to claim 28 or 29 under conditions that allow introduction of the vector into the cell.

33. An isolated or purified host cell comprising the nucleic acid according to anyone of claims 24-27 or the recombinant expression vector according to claim 28 or 29.

34. The host cell according to claim 33, wherein the cell is a human lymphocyte.

35. The host cell according to claim 33 or 34, wherein the cell is selected from the group consisting of a T cell, a natural killer T (NKT) cell, an invariant natural killer T (iNKT) cell, a natural killer (NK) cell, a macrophage, a pluripotent cell, and a multipotent cell.

36. An isolated or purified population of cells comprising the host cell according to any one of claims 33-35.

37. A method of producing the TCR according to any one of claims 1-14, 30, or 31. the polypeptide according to any one of claims 15-18.

30. or 31, or the protein according to any one of claims 19-23, 30, or 31, the method comprising culturing the host cell according to any one of claims 33-35, or the population of host cells according to claim 36, so that the TCR, polypeptide, or protein is produced.

38. A pharmaceutical composition comprising (a) the TCR according to any one of claims 1-14, 30, or 31, the polypeptide according to any one of claims 15-18, 30, or 31, the protein according to any one of claims 19-23, 30, or 31, the nucleic acid according to any one of claims 24-27, the recombinant expression vector according to claim 28 or 29, the host cell according to any one of claims 33-35. or the population of cells according to claim 36 and (b) a pharmaceutically acceptable carrier.

39. A method of detecting the presence of cancer in mammal, the method comprising:(a) contacting a sample comprising cells of the cancer with the TCR according to any one of claims 1-14, 30, or 31, the polypeptide according to any one of claims 15-18, 30, or 31, the protein according to any one of claims 19-23, 30, or 31, the nucleic acid according to any one of claims 24-27. the recombinant expression vector according to claim 28 or 29. the host cell according to any one of claims 33-35, the population of cells according to claim 36, or the pharmaceutical composition of claim 38, thereby forming a complex; and(b) detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal.

40. The TCR according to any one of claims 1-14.

30. or 31, the polypeptide according to any one of claims 15-18, 30, or 31 , the protein according to any one of claims 19-23, 30, or 31, the nucleic acid according to any one of claims 24-27, the recombinant expression vector according to claim 28 or 29, the host cell according to any one of claims 33-35, the population of cells according to claim 36, or the pharmaceutical composition according to claim 38, for use in the inducement of an immune response against cancer in a mammal.

41. The TCR according to any one of claims 1-14. 30, or 31, the polypeptide according to any one of claims 15-18, 30, or 31. the protein according to any one of claims 19-23, 30, or 31, the nucleic acid according to any one of claims 24-27, the recombinant expression vector according to claim 28 or 29, the host cell according to any one of claims 33-35, the population of cells according to claim 36, or the pharmaceutical composition according to claim 38, for use in in the treatment or prevention of cancer in a mammal.

42. The method of claim 39, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for the use of claim 40 or 41, wherein the cancer expresses a mutated human RAS amino acid sequence with a substitution of one or both of (i) glycine at position 12 with aspartic acid, (li) glycine at position 12 with valine, (iiii) glycine at position 13 with aspartic acid or (ii) glutamine at position 61 with arginine, wherein the mutated human RAS amino acid sequence is a mutated human Kirsten rat sarcoma viral oncogene homolog (KRAS), a mutated human Harvey rat sarcoma viral oncogene homolog (HRAS), or a mutated human Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) amino acid sequence, and wherein positions 12, 13 and 61 are defined by reference to the wild-type human KRAS, wild-type human HRAS, or wild-type human NRAS protein, respectively.

43. The method according to claim 39 or 42, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for the use of any one of claims 40-42, wherein the cancer is pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, thyroid cancer, leukemia, melanoma, or prostate cancer.

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