T cell receptors recognizing r175h or y220c mutation in p53

Abstract

To provide an isolated or purified T cell receptor (TCR) having antigen specificity to mutation human p53.SOLUTION: An isolated or purified t cell receptor (TCR) having antigen specificity to human p53R175H or human p53Y220C is provided. A related polypeptide and protein, and related nucleic acid, recombinant expression vector, host cell, cell mass and pharmaceutical composition are also provided. A method for detecting the presence of cancer in a mammal and a method for treating or preventing cancer in a mammal are also provided.SELECTED DRAWING: Figure 1-1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 867,619, filed June 27, 2019, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with federal support by the National Cancer Institute, National Institutes of Health under Project No. BC010985. The federal government has certain rights in this invention.

[0003] Incorporation by Reference of Electronically Filed Materials The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is hereby incorporated by reference in its entirety: One 357,775 byte ASCII (text) file entitled "749338_ST25.txt", dated June 23, 2020. [Background technology]

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

[0005] Brief Summary of the Invention An embodiment of the present invention is a method for the production of human p53 R175H or human p53 Y220CThe present invention provides an isolated or purified T cell receptor (TCR) having antigen specificity for an amino acid sequence of: (1) all of SEQ ID NOs: 3 to 8; (2) all of SEQ ID NOs: 14 to 19; (3) all of SEQ ID NOs: 25 to 30; (4) all of SEQ ID NOs: 36 to 41; (5) all of SEQ ID NOs: 47 to 52; (6) all of SEQ ID NOs: 58 to 63; (7) all of SEQ ID NOs: 69 to 74; (8) all of SEQ ID NOs: 80 to 85; or (9) all of SEQ ID NOs: 131 to 136.

[0006] In one embodiment of the present invention, the TCR is human p53 R175H or human p53 Y220C It has antigen specificity for the amino acid sequence of human p53 R175H The amino acid sequence is SEQ ID NO:2 or SEQ ID NO:96.

[0007] In one embodiment of the present invention, the TCR is human p53 R175H or human p53 Y220C It has antigen specificity for the amino acid sequence of human p53 Y220C The amino acid sequence of is SEQ ID NO:113.

[0008] In one embodiment of the invention, the TCR does not have antigen specificity for the amino acid sequence of wild-type human p53 of SEQ ID NO:95.

[0009] In one embodiment of the invention, the TCR does not have antigen specificity for the amino acid sequence of wild-type human p53 of SEQ ID NO:112.

[0010] Further embodiments of the invention provide related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, cell populations and pharmaceutical compositions related to the TCRs of the invention.

[0011] An embodiment of the present invention is an isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences are set forth in SEQ ID NOs: 9 and 10; 10 and 9; 20 and 21; 21 and 20; 31 and 32; 32 and 31; 42 and 43; 43 and 42; 53 and 54; 54 and 53; 64 and 65; 65 and 64; 75 and 76; 76 and 75; 86 and 87; 87 and 88; 89 and 90; 90 and 91; 91 and 92; 92 and 93; 93 and 94; 94 and 95; 95 and 96; 96 and 97; 97 and 98; 99 and 99; 100 and 100; 100 and 9; 20 and 21; 21 and 20; 31 and 32; 32 and 31; 42 and 43; 43 and 42; 53 and 54; 54 and 53; 64 and 65; 65 and 64; 75 and 76; 76 and 75; 86 and 87; 88 and 89; 99 and 90; 100 and 100; 100 and 9; 20 and 21; 21 and 20; 31 and 32; 32 and 31; 42 and 43; 43 and 42; 7 and 86;137 and 138;138 and 137;142 and 143;143 and 142;144 and 145;145 and 144;146 and 147;147 and 146;148 and 149;149 and 148;150 and 151;151 and 150;152 and 153;153 and 152;154 and 155;155 and 154;156 and 157;157 and 156;159 and 158;158 and 159;178 and 10;10 and 178;181 and 21;21 and 181;184 and 32;32 and 184;187 and 43;43 and 187;190 and 54;54 and 190;193 and 65;65 and 193;196 and 76;76 and 196;199 and 87;87 and 199;137 and 202;202 and 137;9 and 205;205 and 9;20 and 207;207 and 20;31 and 209;209 and 31; Nucleic acids encoding the amino acid sequences of 42 and 211; 211 and 42; 53 and 213; 213 and 53; 64 and 215; 215 and 64; 75 and 217; 217 and 75; 86 and 219; 219 and 86; 137 and 221; 221 and 137; 223 and 202; 202 and 223; 223 and 221; 221 and 223; 20 and 226; 226 and 20; 181 and 226; or 226 and 181 are provided.

[0012] An embodiment of the invention is an isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences are set forth in SEQ ID NOs: 11 and 12; 12 and 11; 22 and 23; 23 and 22; 33 and 34; 34 and 33; 44 and 45; 45 and 44; 55 and 56; 56 and 55; 66 and 67; 67 and 66; 77 and 78; 78 and 77; 88 and 89; 89 and 88;139 and 140;140 and 139;160 and 161;161 and 160;162 and 163;163 and 162;164 and 165;165 and 164;166 and 167;167 and 166;168 and 169;169 and 168;170 and 171;171 and 170;172 and 173;173 and 172;174 and 175;175 and 174;176 and 177;177 and 176;179 and 12;12 and 179;182 and 23;23 and 182;185 and 34;34 and 185;188 and 45;45 and 188;191 and 56;56 and 191;194 and 67;67 and 194;197 and 78;78 and 197;200 and 89;89 and 200;139 and 203;203 and 139;11 and 206;206 and 11;22 and 208;208 and 22;33 and 210;210 and 33 Nucleic acids encoding the amino acid sequences of: ;44 and 212; 212 and 44; 55 and 214; 214 and 55; 66 and 216; 216 and 66; 77 and 218; 218 and 77; 88 and 220; 220 and 88; 139 and 222; 222 and 139; 224 and 203; 203 and 224; 224 and 222; 222 and 224; 22 and 227; 227 and 22; 182 and 227; or 227 and 182 are provided.

[0013] In one embodiment of the invention, the isolated or purified nucleic acid further comprises a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, said third nucleotide sequence encoding a cleavable linker peptide.

[0014] In one embodiment of the invention, the cleavable linker peptide is the amino acid sequence of SEQ ID NO:94. It contains the amino acid sequence.

[0015] In one embodiment of the invention, the isolated or purified nucleic acid encodes an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 24, 35, 46, 57, 68, 79, 90, 141, 180, 183, 186, 189, 192, 195, 198, 201, 204, 225, 228, and 229.

[0016] In one embodiment of the invention, the recombinant expression vector is a transposon-based vector or a lentiviral vector.

[0017] Another embodiment of the invention provides an isolated or purified TCR, polypeptide, or protein encoded by any of the nucleic acids or vectors described herein.

[0018] Another embodiment of the invention provides an isolated or purified TCR, polypeptide, or protein resulting from expression in a cell of any of the nucleic acids or vectors described herein.

[0019] Another embodiment of the invention provides a method of producing a host cell expressing a TCR having antigen specificity for a peptide of SEQ ID NO: 2, 96 or 113, comprising contacting the cell with any of the vectors described herein under conditions that allow for introduction of the vector into the cell.

[0020] Another embodiment of the invention provides an isolated or purified host cell comprising any of the nucleic acids or recombinant expression vectors described herein.

[0021] In one embodiment of the invention, the host cell is a human lymphocyte.

[0022] In one embodiment 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, and a natural killer (NK) cell.

[0023] Another embodiment of the invention provides a method of producing any of the TCRs, polypeptides or proteins described herein, comprising culturing any of the host cells, or host cell populations described herein, such that the TCR, polypeptide or protein is produced.

[0024] Another embodiment of the invention provides any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions described herein for use in inducing an immune response against cancer in a mammal. In one embodiment, the invention provides a method of inducing an immune response against cancer in a mammal comprising administering any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions described herein.

[0025] Yet further embodiments of the invention provide methods of detecting the presence of cancer in a mammal, and methods of treating or preventing cancer in a mammal. In one embodiment, the invention provides a method of treating or preventing cancer in a mammal comprising administering any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, cell populations, or pharmaceutical compositions described herein. In one embodiment, the cancer is cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

[0026] In one embodiment of the invention, the cancer is known to contain a R175H mutation or a Y220C mutation in human p53. [Brief description of the drawings]

[0027] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1-1]FIG. 1A is a schematic diagram showing the experimental design (parent gates are lymphocytes → single cells → live (PI negative) → CD3+ (T cells)). [Figure 1-2] 1B-1C are graphs showing the percentage of 4-1BB positive cells detected after IVS (TP53-TMG-IVS (B) or p53-LP-IVS (C)) and 4-1BB / OX40 enrichment. Cultures considered positive are shown in bold. Responses to mutant TP53 (TMG (black circles); LP (black squares)) and wild-type (WT) counterparts (TMG (white circles); LP (white squares)) are shown. [Figure 1-3] FIG. 1D shows representative flow cytometry plots from 4141-CD8 TP53-TMG-IVS cultures following co-culture of WT or mutated (MUT) TP53 tandem minigene (TMG) with electroporated autologous antigen-presenting cells. [Figure 1-4] Figure 1E shows interferon gamma secretion (left axis) or upregulation of 4-1BB (right axis) measured after antigen-experienced CD4 T cells were sorted and stimulated in vitro with immature dendritic cells electroporated with mutant TP53-TMG. Cultures were then co-cultured with immature dendritic cells electroporated with mutant TP53-TMG, and the next day 4-1BB+ and / or OX40+ cells were sorted and amplified by a rapid amplification protocol. After 12-14 days of expansion, cultures (4285-CD4 TP53-TMG-IVS) were tested for specificity for the p53-R175H neoantigen by interferon gamma ELISPOT (left axis) or for upregulation of 4-1BB by flow cytometry (right axis). [Figure 1-5]FIG. IF is a graph showing interferon-gamma secretion measured by ELISA on supernatants of co-cultures of 4285-CD4 TP53-TMG-IVS with peptide-pulsed autologous antigen-presenting cells. 4285-CD4 TP53-TMG-IVS cultures were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either the 25 amino acid long WT (open circles) or mutant (closed squares) p53-R175 peptide. After overnight incubation, co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are mean ± SEM (n=3 technical replicates). [Figure 2-1] Figure 2A is a graph showing clonality of the total population, which is a normalized estimate of sample diversity (numbers closer to 1 are less diverse). TCRB sequencing was performed on PBL before and after amplification by IVS and 4-1BB / OX40 enrichment by either LP or TMG. Cultures with confirmed p53 neoantigen responses are highlighted with an asterisk. Figure 2B is a graph showing the maximum frequency of productive and unique CDR3B from each population. TCRB sequencing was performed on PBL before and after amplification by IVS and 4-1BB / OX40 enrichment by either LP or TMG. Cultures with confirmed p53 neoantigen responses are highlighted with an asterisk. [Figure 2-2] FIG. 2C is a graph showing the results of an experiment in which non-transduced T cells (negative control for TCR-transduced T cells) were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed by ELISA for interferon gamma secretion. Data are means ± SEM (n=3). FIG. 2D is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR1 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed by ELISA for interferon gamma secretion. Data are means ± SEM (n=3). [Figure 2-3] FIG. 2E is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR2 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are means ± SEM (n=3). FIG. 2F is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR3 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are means ± SEM (n=3). [Figure 2-4] FIG. 2G is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR5 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are means ± SEM (n=3). FIG. 2H is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR6 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are means ± SEM (n=3). [Figure 2-5]FIG. 2I is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR7 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are means ± SEM (n=3). FIG. 2J is a graph showing the results of an experiment in which T cells transduced with 4285-PBL-TCR9 were co-cultured with immature dendritic cells pulsed with decreasing concentrations of either WT (open circles) or mutated (closed squares) p53-R175 peptides, 25 amino acids long. After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are means ± SEM (n=3). [Figure 2-6] Figures 2K-2L are graphs showing the percentage of TCRB clonotypes measured after tracking CDR3B with known specificity for mutant TP53 before (PBL) and after (IVS / enrichment) the IVS and 4-1BB enrichment protocol. P53R175H-specific clonotypes are shown in K. P53R248W-specific clonotypes are shown in L. [Figure 3-1] FIG. 3A is a graph showing the percentage of 4-1BB positive cells measured after transfection of COS7 monkey cell lines with the indicated HLA and pulsing with the indicated shortest p53 peptides (WT (white bars); mutant (black bars)). Results from 4141-CD8 TP53-TMG-IVS cultures are shown. TMG-wtR175 had mutated TP53 at all positions except R175H. The sequence HMTEVVRRC is SEQ ID NO: 95. The sequence HMTEVVRHC is SEQ ID NO: 96. FIG. 3B is a graph showing the amount of IFN-γ measured after transfection of COS7 monkey cell lines with the indicated HLA and pulsing with the indicated shortest p53 peptides (WT (white bars); mutant (black bars)). Results from 4266-CD8 TP53-TMG-IVS cultures are shown. The sequence SSCMGGMNRR is SEQ ID NO: 97. The sequence SSCMGGMNWR is SEQ ID NO: 98. [Figure 3-2]FIG. 3C is a graph showing the results of an experiment in which the COS7 monkey tumor cell line was transfected with HLA plasmid DNA corresponding to the haplotype of patient 4285 and either WT TP53 TMG (only the R175H position) (WT-R175-TMG; open bars) or mutated TP53-TMG containing the p53-R175H neoantigen (closed bars). The next day, 4285-CD4 TP53-TMG-IVS cultures were added and co-cultured. After overnight incubation, co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are mean ± SEM (n=3). [Figure 3-3] FIG. 3D shows flow cytometry plots (4266-CD8 on the left, 4141-CD8 on the right) showing upregulation of 4-1BB in CD8+ T cells from TP53-TMG-IVS cultures after coculture of TC#4266 (autologous xenograft from patient 4266; A*68:01; p53R248W) with Saos2 cells overexpressing the full-length p53R175H gene (A*02:01). [Figure 4] Figure 4 is a graph showing the results of an experiment in which the COS7 monkey tumor cell line was transfected with DRA1*01:01:01 and DRB1*13:01:01 and either no relationship (white bars), WT TP53 TMG (only the R175H position) (WT-R175-TMG; grey bars) or mutated TP53-TMG containing the p53-R175H neoantigen (black bars). The next day, 4285-PBL-TCR transduced or non-transduced T cells were added and co-cultured. After overnight incubation, co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are mean ± SEM (n=3). [Diagram 5]Figure 5 is a graph showing the results of an experiment in which CD8+ T cells were sorted from 4259-F1 tumor fragment cultures, followed by preparation of T cell clones by limiting dilution. 24 cultures were co-cultured with T2 tumor cells (HLA-A*02:01) pulsed with DMSO (peptide vehicle), WT p53-Y220 peptide, or MUT p53-Y220C peptide. After overnight incubation, cells were stained for CD3, CD8, and 4-1BB and then analyzed by flow cytometry. The frequency of CD8+4-1BB+ T cells from the cultures is shown. [Figure 6] FIG. 6 shows that 4259-F1-TCR was transduced into donor peripheral blood T cells followed by decreasing concentrations of the WT p53-Y220 peptide.

[0028] [ka]

[0029] or MUT p53-Y220C peptide

[0030] [ka]

[0031] T2 tumor cells pulsed with either HLA-A * 02:01). After overnight incubation, co-culture supernatants were analyzed for interferon gamma secretion by ELISA. Data are mean ± SEM (n=3). [Figure 7]FIG. 7 is a graph showing the results of an experiment in which T cells either not expressing TCR (non-transduced), expressing p53-R175H-specific TCR, or expressing 4259-F1-TCR were co-cultured with tumor cells (either with or without expression of HLA-A*02:01, p53-R175H, or p53-Y220C). After overnight incubation, cells were stained for CD3, CD8, and 4-1BB and then analyzed by flow cytometry. The frequency of CD8+4-1BB+ T cells from the cultures is shown. Data are mean±SEM (n=3). [Figure 8] Figure 8 shows an alignment of the amino acid sequences of the nine p53 splice variants: SP|P04637|P53_HUMAN (SEQ ID NO: 1); SP|P04637-2|P53_HUMAN (SEQ ID NO: 114); SP|P04637-3|P53_HUMAN (SEQ ID NO: 115); SP|P04637-4|P53_HUMAN (SEQ ID NO: 116); SP|P04637-5|P53_HUMAN (SEQ ID NO: 117); SP|P04637-6|P53_HUMAN (SEQ ID NO: 118); SP|P04637-7|P53_HUMAN (SEQ ID NO: 119); SP|P04637-8|P53_HUMAN (SEQ ID NO: 120); and SP|P04637-9|P53_HUMAN (SEQ ID NO: 121). [Figure 9] 9 shows an alignment of a partial amino acid sequence of the TRBV7-9*03 sequence with that of TRBV7-9*01. "L36092|TRBV7-9*01|Homo" is SEQ ID NO: 129. "AF009663|TRBV7-9*03|Homo" is SEQ ID NO: 130. [Figure 10]10 is a graph showing the percentage of 4-1BB positive cells (% of CD8+) (right y-axis; black bars) and IFN-γ (spots per 2×104 cells) (left y-axis; hatched bars) measured after co-culture of TILs from patient 4141 (fragment culture 12) with autologous APC transfected with TMG encoding an irrelevant mutation (TMG-IRR), a WT p53 sequence (TP53-wt-TMG), or a mutated p53 sequence containing R175H (TP53-mut-TMG). Medium alone, and PMA and ionomycin were negative and positive controls, respectively. [Figure 11] FIG. 11 is a graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after coculture of TILs from patient 4141 (fragment culture 12) with Cos7 cells cotransfected with the indicated HLA alleles and either no additional genes (HLA only, open bars), WT TP53 TMG (grey hatched bars), or mutant TP53 TMG containing the p53-R175H sequence (black bars). [Figure 12] Figure 12 is a graph showing the concentration of IFN-γ (pg / mL) measured after co-culture of mock (no TCR) or 4141-TCR1a2 expressing T cells with T2 tumor cells (expressing HLA-A*02). T2 cells were pulsed with peptide vehicle (DMSO; grey bars) or purified (>95% by HPLC) peptides consisting of WT p53-R175 peptide (hatched grey bars) or mutant p53-R175H peptide (black bars). Media only (white bars), and PMA and ionomycin (checkered bars) were negative and positive controls, respectively. Data are mean ± SEM (n=3). [Figure 13]Figure 13 is a graph showing the percentage of cells positive for expression of one of the indicated markers after co-culture of T cells expressing 4141-TCR1a2 with Saos2 cells (p53-NULL and HLA-A*02:01+) either unmanipulated (unshaded bars) or engineered to overexpress full-length p53-R175H protein (shaded bars). Data are mean ± SEM (n=3). For statistical analysis, a two-tailed Student's t-test was performed for each cytokine between the two cell lines (***p<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Tumor protein P53 (also called "TP53" or "p53") acts as a tumor suppressor, for example, by controlling cell division. The p53 protein is located in the nucleus of the cell, where it binds directly to DNA. When DNA is damaged, the p53 protein is involved in the decision to repair the DNA or to cause the damaged cell to undergo apoptosis. If the DNA can be repaired, p53 activates other genes to repair the damage. If the DNA cannot be repaired, the p53 protein stops the cell from dividing and signals the cell to undergo apoptosis. By stopping the division of cells with mutated or damaged DNA, p53 helps to prevent the development of tumors. WT (normal) full-length p53 comprises the amino acid sequence of SEQ ID NO:1.

[0033] Mutations in p53 protein may reduce or eliminate the tumor suppressor function of p53 protein.Alternatively or additionally, p53 mutations may be gain-of-function mutations by interfering with WTp53 in a dominant-negative manner.Mutant p53 protein may be expressed in any of a variety of human cancers, such as cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

[0034] An embodiment of the present invention provides an isolated or purified T cell receptor (TCR) having antigen specificity for mutated human p53 (hereinafter "mutated p53"). Hereinafter, reference to "TCR" also refers to functional portions and functional variants of the TCR, unless otherwise specified. Mutations of p53 are defined herein by reference to the amino acid sequence of full-length WT p53 (SEQ ID NO: 1). Mutations of p53 are described herein by reference to the amino acid residue present at a particular position, followed by the position number, followed by the amino acid that replaces that residue in the particular mutation under consideration. An amino acid sequence of p53 (e.g., a p53 peptide) may contain less than all of the amino acid residues of the full-length WT p53 protein. Thus, position numbers are defined herein by reference to the WT full-length p53 protein (i.e., SEQ ID NO: 1), with the understanding that the actual positions of the corresponding residues in a particular example of a p53 amino acid sequence may vary. As the positions are defined by SEQ ID NO:1, the term "R175" refers to the arginine present at position 175 of SEQ ID NO:1, "R175H" indicates that the arginine present at position 175 of SEQ ID NO:1 has been replaced with a histidine, while "Y220C" indicates that the tyrosine present at position 220 of SEQ ID NO:1 has been replaced with a cysteine. For example, a specific example of an amino acid sequence for p53 is, e.g., YKQSQHMTEVVR R CPHHERCSDSDG (SEQ ID NO:110) (an exemplary WT p53 peptide corresponding to consecutive amino acid residues 163-187 of SEQ ID NO:1), "R175H" refers to the substitution of the underlined arginine in SEQ ID NO:110 with histidine, even though the actual position of the underlined arginine in SEQ ID NO:110 is 13. Hereinafter, a human p53 amino acid sequence having an R175H mutation will be referred to as "R175H" or "p53 R175H Hereinafter, the amino acid sequence of human p53 having the Y220C mutation will be referred to as "Y220C" or "p53 Y220C As used herein, "mutated p53" refers to human p53 R175H or human p53 Y220C Refers to...

[0035] There are nine known splice variants of p53. The p53 mutations described herein are conserved across all nine p53 splice variants. An alignment of the nine p53 splice variants is shown in FIG. 8. Thus, the TCRs of the present invention may have antigen specificity for any mutant p53 amino acid sequence described herein encoded by any of the nine p53 splice variants. Since the positions are as defined by SEQ ID NO:1, the actual positions of the amino acid sequence of a particular splice variant of p53 are defined relative to the corresponding positions in SEQ ID NO:1, and the positions defined by SEQ ID NO:1 may differ from the actual positions in a particular splice variant. Thus, for example, a mutation refers to a substitution of an amino acid residue in the amino acid sequence of a particular splice variant of p53 that corresponds to the indicated positions of the 393 amino acid sequence of SEQ ID NO:1, with the understanding that the actual positions in a splice variant may differ.

[0036] In one embodiment of the present invention, the TCR has antigen specificity for human p53 with a mutation at position 175 as defined by SEQ ID NO: 1. The p53 mutation at position 175 can be any missense mutation. Thus, the mutation at position 175 can be a substitution of the naturally occurring (WT) arginine residue at position 175 with any amino acid residue other than arginine. In one embodiment of the present invention, the TCR has antigen specificity for human p53 with an R175H mutation. For example, the TCR of the present invention can be a human p53 with an R175H mutation, H CPHHER (SEQ ID NO: 2), HMTEVVR H C (SEQ ID NO:96), KQSQHMTEVVR H CPH(sequence No. 100), QSQHMTEVVR H CPHH (SEQ ID NO: 101), SQHMTEVVR H CPHHE (SEQ ID NO: 102), QHMTEVVR H CPHHER (SEQ ID NO: 103), HMTEVVR H CPHHERC (SEQ ID NO: 104), MTEVVR H CPHHERCS (SEQ ID NO: 105), TEVVRH CPHHERCSD (SEQ ID NO: 106), EVVR H CPHHERCSDS (SEQ ID NO: 107), VVR H CPHHERCSDSD (SEQ ID NO: 108), VR H CPHHERCSDSDG (SEQ ID NO: 109), YKQSQHMTEVVR H The antigen may have antigen specificity for one or more mutant p53 amino acid sequences selected from the group consisting of: CPHHERCSDSDG (SEQ ID NO:111).

[0037] In one embodiment of the invention, the TCR has antigen specificity for human p53 with a mutation at position 220 as defined by SEQ ID NO: 1. The p53 mutation at position 220 can be any missense mutation. Thus, the mutation at position 220 can be a substitution of the native (WT) tyrosine residue present at position 220 with any amino acid residue other than tyrosine. In one embodiment of the invention, the TCR has antigen specificity for human p53 with a Y220C mutation. For example, the TCR of the invention comprises:

[0038] [ka]

[0039] The p53 antibody may have antigenic specificity for the mutated p53 amino acid sequence.

[0040] In an embodiment of the present invention, the TCR of the present invention can recognize mutant p53 in an HLA (human leukocyte antigen) molecule-dependent manner. "HLA molecule-dependent manner" as used herein means that the TCR induces an immune response when it binds to mutant p53 in the context of the HLA molecule expressed in the patient from which the TCR is isolated. The TCR of the present invention can recognize mutant p53 presented by applicable HLA molecules, and can bind to HLA molecules in addition to mutant p53.

[0041] In one embodiment of the present invention, the TCR of the present invention can recognize R175H presented by HLA class II molecules. In this regard, the TCR can induce an immune response upon binding to R175H in the context of HLA class II molecules. The TCR of the present invention can recognize R175H presented by HLA class II molecules and can bind to HLA class II molecules in addition to R175H.

[0042] In one embodiment of the present invention, the HLA class II molecule is an HLA-DR heterodimer. The HLA-DR heterodimer is a cell surface receptor that comprises an α chain and a β chain. The HLA-DR α chain is encoded by the HLA-DRA gene. In one embodiment, the alpha chain of the HLA class II molecule is HLA-DRA1. * It is expressed by the 01:01:01 allele. The HLA-DRβ chain is encoded by the HLA-DRB1 gene, the HLA-DRB3 gene, the HLA-DRB4 gene, or the HLA-DRB5 gene. Examples of molecules encoded by the HLA-DRB1 gene may include, but are not limited to, HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4, HLA-DR5, HLA-DR6, HLA-DR7, HLA-DR8, HLA-DR9, HLA-DR10, HLA-DR11, HLA-DR12, HLA-DR13, 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 one embodiment of the present invention, the HLA class II molecule is an HLA-DRB1:HLA-DRA heterodimer. The beta chain of the HLA class II molecule is HLA-DRB1:HLA-DRA heterodimer. * 13:01, HLA -DRB1 * 13:02, HLA-DRB1 * 13:03, HLA-DRB1 * 13:04, HLA-DRB1 * 13:05, HLA-DRB1 * 13:06, HLA-DRB1* 13:07, HLA-DRB1 * 13:08, HLA-DRB1 * 13:09, or HLA-DRB1 * In a particularly preferred embodiment, the beta chain of the HLA class II molecule is expressed by the HLA-DRB1 * It is expressed by the 13:01 allele.

[0043] In one embodiment of the invention, one of the TCRs of the invention is capable of recognizing Y220C presented by HLA class I molecules. In this regard, the TCR may elicit an immune response upon binding to Y220C in the context of an HLA class I molecule. The TCR of the invention is capable of recognizing Y220C presented by HLA class I molecules and may bind to HLA class I molecules in addition to Y220C.

[0044] In one embodiment of the present invention, the HLA class I molecule is an HLA-A molecule. An HLA-A molecule is a heterodimer of an alpha chain and β2 microglobulin. The HLA-A alpha chain may be encoded by the HLA-A gene. β2 microglobulin non-covalently binds to the alpha1, alpha2, and alpha3 domains of the alpha chain to form the HLA-A complex. The HLA-A molecule may be any HLA-A molecule. In one embodiment of the present invention, the HLA class I molecule is an HLA-A2 molecule. The HLA-A2 molecule may be any HLA-A2 molecule. An example of an HLA-A2 molecule is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:05, HLA-A * 02:06, HLA-A * 02:07, or HLA-A * Preferably, the HLA class I molecule is HLA-A or HLA-C. * 02:01 molecule.

[0045] The TCRs of the invention, including when expressed in cells used for adoptive cell transfer, may provide any one or more of a number of advantages. Mutant p53 is expressed in cancer cells and not in normal non-cancer cells. Without being bound to a particular theory or mechanism, it is believed that the TCRs of the invention advantageously target and thereby reduce the destruction of cancer cells while simultaneously minimizing or eliminating the destruction of normal non-cancer cells, e.g., by minimizing or eliminating toxicity. Furthermore, the TCRs of the invention may advantageously successfully treat or prevent mutant p53 positive cancers that are unresponsive to other types of treatment, e.g., chemotherapy, surgery, or radiation. Furthermore, the TCRs of the invention may be able to recognize mutant p53 with high avidity, which may confer the ability to recognize unengineered tumor cells (e.g., tumor cells that have not been treated with interferon (IFN)-γ, transfected with vectors encoding one or both of mutant p53 and the applicable HLA molecule, pulsed with p53 peptides carrying p53 mutations, or combinations thereof). Approximately half of all tumors harbor p53 mutations, of which about half are missense mutations. The R175H mutation is expressed in approximately 4.5% of all cancers and is associated with HLA-DRB1 * The 13:01 allele is expressed in approximately 15% of the American population. The Y220C mutation occurs in approximately 1.5% of all cancers and is associated with HLA-A * The 02:01 allele is expressed in about 40% to about 50% of the American population. The R175H and Y220C mutations occur in many cancer histologies, suggesting that a diverse group of patients may benefit from the TCRs of the present invention. Thus, the TCRs of the present invention can increase the number of patients who may be eligible for immunotherapy treatment.

[0046] The term "antigen specificity" as used herein means that the TCR can specifically bind to and immunologically recognize mutant p53 with high binding activity. For example, (a) a low concentration of mutant p53 peptide (e.g., about 0.05 ng / mL to about 5 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, or values ​​above that) (b) a nucleotide sequence encoding mutant p53 is introduced into the target cells so that the target cells express mutant p53, and the target cells express TCR. 4 ~Approx. 1×10 5 If a T cell secretes at least about 200 pg / mL or more (e.g., 200 pg / mL or more, 300 pg / mL or more, 400 pg / mL or more, 500 pg / mL or more, 600 pg / mL or more, 700 pg / mL or more, 1000 pg / mL or more, 5,000 pg / mL or more, 7,000 pg / mL or more, 10,000 pg / mL or more, 20,000 pg / mL or more, or a range defined by any two of the above values), the TCR may be considered to have "antigen specificity" for mutant p53. Cells expressing the TCR of the present invention can also secrete IFN-γ when co-cultured with antigen-negative applicable HLA molecule-positive target cells pulsed with a higher concentration of mutant p53 peptide.

[0047] Alternatively or additionally, a TCR may be considered to have "antigen specificity" for mutant p53 if T cells expressing the TCR secrete at least twice as much IFN-γ when co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with a low concentration of mutant p53 peptide or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding mutant p53 has been introduced such that the target cells express mutant p53, compared to the amount of IFN-γ expressed in a negative control. A 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 having a sequence different from the mutated p53 peptide) or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding an irrelevant peptide has been introduced so that the target cells express the irrelevant peptide, or (ii) non-transduced T cells (e.g., from PBMCs not expressing the TCR) co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with the same concentration of the mutated p53 peptide or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding mutated p53 has been introduced so that the target cells express mutated p53. IFN-γ secretion can be measured by methods known in the art, for example, enzyme-linked immunosorbent assay (ELISA).

[0048] Alternatively or additionally, a TCR may be considered to have "antigen specificity" for mutant p53 if the number of T cells expressing a TCR secreting IFN-γ is at least twice as high when co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with a low concentration of mutant p53 peptide or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding mutant p53 has been introduced so that the target cells express mutant p53, compared to the number of negative control T cells secreting IFN-γ. The concentration of peptide and negative control may be as described herein for other aspects of the invention. The number of cells secreting IFN-γ can be measured by methods known in the art, for example, enzyme-linked immunospot (ELISPOT) assay.

[0049] Alternatively or additionally, a TCR may be considered to have "antigen specificity" for mutant p53 if at least twice as many spots are detected by ELISPOT on T cells expressing the TCR when co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with a low concentration of mutant p53 peptide or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding mutant p53 has been introduced such that the target cells express mutant p53, compared to the number of spots detected by ELISPOT on negative control T cells co-cultured with the same target cells. The peptide concentrations and negative controls may be as described herein for other aspects of the invention.

[0050] Alternatively or additionally, a TCR may be considered to have "antigen specificity" for mutant p53 if more than about 50 spots are detected by ELISPOT for T cells expressing the TCR when co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with low concentrations of mutant p53 peptide or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding mutant p53 has been introduced such that the target cells express mutant p53. The concentrations of peptides may be as described herein for other aspects of the invention.

[0051] Alternatively, or in addition, a TCR may be considered to have "antigen specificity" for mutant p53 if a T cell expressing the TCR upregulates expression of one or both of 4-1BB and OX40, as measured, for example, by flow cytometry, following stimulation with a target cell expressing mutant p53.

[0052] One embodiment of the present invention provides a TCR comprising two polypeptides (i.e., polypeptide chains), such as, for example, a TCR alpha (α) chain, a TCR beta (β) chain, a TCR gamma (γ) chain, a TCR delta (δ) chain, or a combination thereof. The polypeptides of the TCR of the present invention may comprise any amino acid sequence, so long as the TCR has antigen specificity for mutant p53.

[0053] In an embodiment of the invention, a TCR comprises two polypeptide chains, each comprising a variable region comprising TCR complementarity determining region (CDR) 1, CDR2, and CDR3. In an embodiment of the invention, a TCR comprises a first polypeptide chain comprising CDR1 of the alpha chain (CDR1α), CDR2 of the alpha chain (CDR2α), and CDR3 of the alpha chain (CDR3α), and a second polypeptide chain comprising CDR1 of the beta chain (CDR1β), CDR2 of the beta chain (CDR2β), and CDR3 of the beta chain (CDR3β). In an embodiment of the invention, the TCR comprises the amino acid sequence of (1) all of SEQ ID NOs: 3-8; (2) all of SEQ ID NOs: 14-19; (3) all of SEQ ID NOs: 25-30; (4) all of SEQ ID NOs: 36-41; (5) all of SEQ ID NOs: 47-52; (6) all of SEQ ID NOs: 58-63; (7) all of SEQ ID NOs: 69-74; (8) all of SEQ ID NOs: 80-85; or (9) all of SEQ ID NOs: 131-136. Each one of the collections of nine amino acid sequences described above in this paragraph represents six CDR regions of each of nine different TCRs having antigen specificity for mutant human p53. The six amino acid sequences in each collection correspond to CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β, respectively.

[0054] In an embodiment of the invention, the TCR comprises an alpha chain variable region amino acid sequence and a beta chain variable region amino acid sequence which together comprise one of the above collections of CDRs. In this regard, the TCR may, for example, comprise the amino acid sequence of any one of SEQ ID NOs: 9, 10, 20, 21, 31, 32, 42, 43, 53, 54, 64, 65, 75, 76, 86, 87, 137, 138, 142-159, 178, 181, 184, 187, 190, 193, 196, 199, 202, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, and 226. For example, the TCR may be: (1) both SEQ ID NOs:9 and 10; (2) both SEQ ID NOs:20 and 21; (3) both SEQ ID NOs:31 and 32; (4) both SEQ ID NOs:42 and 43; (5) both SEQ ID NOs:53 and 54; (6) both SEQ ID NOs:64 and 65; (7) both SEQ ID NOs:75 and 76; (8) both SEQ ID NOs:86 and 87; (9) both SEQ ID NOs:137 and 138; (10) both SEQ ID NOs:142 and 143; (11) both SEQ ID NOs:144 and 145; (12) both SEQ ID NOs:146 and 147; (13) both SEQ ID NOs:148 and 149; (14) both SEQ ID NOs:150 and 151; (15) both SEQ ID NOs:152 and 153; (16) both SEQ ID NOs:154 and 155; (17) both SEQ ID NOs:156 and 157; (18) SEQ ID NOs:159 and 158 (19) both SEQ ID NOs:178 and 10; (20) both SEQ ID NOs:181 and 21; (21) both SEQ ID NOs:184 and 32; (22) both SEQ ID NOs:187 and 43; (23) both SEQ ID NOs:190 and 54; (24) both SEQ ID NOs:193 and 65; (25) both SEQ ID NOs:196 and 76; (26) both SEQ ID NOs:199 and 87; (27) both SEQ ID NOs:137 and 202; (28) both SEQ ID NOs:9 and 205; (29) both SEQ ID NOs:20 and 207; (30) SEQ ID NOs: (31) both SEQ ID NOs: 31 and 209; (31) both SEQ ID NOs: 42 and 211; (32) both SEQ ID NOs: 53 and 213; (33) both SEQ ID NOs: 64 and 215; (34) both SEQ ID NOs: 75 and 217; (35) both SEQ ID NOs: 86 and 219; (36) both SEQ ID NOs: 137 and 221; (37) both SEQ ID NOs: 223 and 202; (38) both SEQ ID NOs: 223 and 221; (39) both SEQ ID NOs: 20 and 226; or (40) both SEQ ID NOs: 181 and 226. Each one of the collections of 47 amino acid sequences described above in this paragraph represents two variable regions of each of 47 different TCRs having antigen specificity for mutant human p53. The two amino acid sequences in each collection correspond to the variable regions of the α chain and the variable regions of the β chain of the TCR, respectively.

[0055] The TCRs of the invention may further comprise a constant region. The constant region may be from any suitable species, e.g., human or mouse. In an embodiment of the invention, the TCR further comprises a murine constant region. As used herein, the terms "murine" or "human" when referring to a TCR or any component of a TCR described herein (e.g., complementarity determining regions (CDRs), variable regions, constant regions, alpha chain, and / or beta chain), refer to a TCR (or component thereof) of murine or human origin, respectively, i.e., a TCR (or component thereof) that originates from or was once expressed by a murine T cell or a human T cell, respectively. In an embodiment of the invention, the TCR may comprise a murine α chain constant region and a murine β chain constant region. The murine α chain constant region may be modified or unmodified. The modified murine α chain constant region may be, for example, cysteine ​​substituted, LVL modified, or both cysteine ​​substituted and LVL modified, e.g., as described in U.S. Pat. No. 10,174,098. The mouse β chain constant region may be modified or unmodified. The modified mouse β chain constant region may be, for example, cysteine ​​substituted, for example, as described in U.S. Patent No. 10,174,098. In an embodiment of the invention, the TCR comprises a cysteine ​​substituted LVL modified mouse α chain constant region comprising the amino acid sequence of SEQ ID NO: 91 or 92. In an embodiment of the invention, the TCR comprises a cysteine ​​substituted mouse β chain constant region comprising the amino acid sequence of SEQ ID NO: 93.

[0056] In an embodiment of the present invention, the TCR of the present invention may comprise a TCR α chain and a TCR β chain. The TCR α chain may comprise an α chain variable region and an α chain constant region. Such an α chain may pair with any TCR β chain. The β chain may comprise a β chain variable region and a β chain constant region.

[0057] In some embodiments, the amino acid sequences of any of the alpha and / or beta chains disclosed herein further comprise the amino acid sequence RAKR (SEQ ID NO: 230) at the C-terminus.

[0058] In an embodiment of the invention, the TCR comprises the amino acid sequence of any one of SEQ ID NOs: 11, 12, 22, 23, 33, 34, 44, 45, 55, 56, 66, 67, 77, 78, 88, 89, 139, 140, 160-177, 179, 182, 185, 188, 191, 194, 197, 200, 203, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, and 227. For example, the TCR may be: (1) both of SEQ ID NOs: 11 and 12; (2) both of SEQ ID NOs: 22 and 23; (3) both of SEQ ID NOs: 33 and 34; (4) both of SEQ ID NOs: 44 and 45; (5) both of SEQ ID NOs: 55 and 56; (6) both of SEQ ID NOs: 66 and 67; (7) both of SEQ ID NOs: 77 and 78; (8) both of SEQ ID NOs: (10) both SEQ ID NOs:160 and 161; (11) both SEQ ID NOs:162 and 163; (12) both SEQ ID NOs:164 and 165; (13) both SEQ ID NOs:166 and 167; (14) both SEQ ID NOs:168 and 169; (15) both SEQ ID NOs:170 and 171; (16) both SEQ ID NOs:172 and 173; (17) both SEQ ID NOs:174 and 175; (18) both SEQ ID NOs:176 and 177; (19) both SEQ ID NOs:179 and 12; (20) both SEQ ID NOs:182 and 23; (21) both SEQ ID NOs:185 and 34; (22) both SEQ ID NOs:188 and 45; (23) both SEQ ID NOs:191 and 56; (24) both SEQ ID NOs:194 and 67 (25) both SEQ ID NOs:197 and 78; (26) both SEQ ID NOs:200 and 89; (27) both SEQ ID NOs:139 and 203; (28) both SEQ ID NOs:11 and 206; (29) both SEQ ID NOs:22 and 208; (30) both SEQ ID NOs:33 and 210; (31) both SEQ ID NOs:44 and 212; (32) both SEQ ID NOs:55 and 214; (33) both SEQ ID NOs:66 and 216; (34) both SEQ ID NOs:77 and 218; (35) both SEQ ID NOs:88 and 220; (36) both SEQ ID NOs:139 and 222; (37) both SEQ ID NOs:224 and 203; (38) both SEQ ID NOs:224 and 222; (39) both SEQ ID NOs:22 and 227; or (40) both SEQ ID NOs:182 and 227. Each one of the collections of amino acid sequences described above in this paragraph represents a respective α-chain and β-chain of a different TCR having antigen specificity for mutant human p53. The two amino acid sequences in each collection correspond to the α-chain and β-chain of the TCR, respectively.

[0059] The functional variants of the TCRs of the present invention described herein are included within the scope of the present invention. The term "functional variant" as used herein refers to a TCR, polypeptide, or protein that has substantial or significant sequence identity or similarity to a parent TCR, polypeptide, or protein, and the functional variant retains the biological activity of the TCR, polypeptide, or protein of which it is a variant. Functional variants include variants of the TCRs, polypeptides, or proteins described herein (parent TCRs, polypeptides, or proteins) that retain, for example, to a similar extent, the same extent, or to a greater extent than the parent TCR, polypeptide, or protein, the antigen specificity of the parent TCR, or the ability to specifically bind to mutant p53 to which the parent polypeptide or protein specifically binds. In relation to a parent TCR, polypeptide or protein, a functional variant may, for example, be at least about 30%, at least about 50%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more identical in amino acid sequence to the parent TCR, polypeptide or protein, respectively.

[0060] A functional variant may, for example, comprise an amino acid sequence of a 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 an amino acid having particular physical and / or chemical properties is exchanged for another amino acid having the same chemical or physical properties. For example, a conservative amino acid substitution may be the substitution of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), an amino acid having a non-polar side chain with another amino acid having a non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid with another basic amino acid (Lys, Arg, etc.), an amino acid having a polar side chain with another amino acid having a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0061] Alternatively, or in addition, a functional variant may comprise the amino acid sequence of a parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. Preferably, the conservative amino acid substitutions do not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitutions enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide or protein.

[0062] A TCR, polypeptide, or protein may consist essentially of the specified amino acid sequence(s) described herein, such that other components of the TCR, polypeptide, or protein, e.g., other amino acids, do not substantially alter the biological activity of the TCR, polypeptide, or protein.

[0063] Also provided by the invention are polypeptides comprising a functional portion of any of the TCRs described herein. The term "polypeptide" as used herein includes oligopeptides and refers to a single chain of amino acids linked by one or more peptide bonds.

[0064] In the context of the polypeptides of the invention, a functional portion may be any portion comprising consecutive amino acids of the TCR of which it is a part, so long as it specifically binds to mutant p53. The term "functional portion", when used in the context of a TCR, refers to any portion or fragment of the TCR of the invention that retains the biological activity of the TCR of which it is a part (the parent TCR). A functional portion encompasses, for example, a portion of the TCR that retains the ability to specifically bind to mutant p53 or detect, treat, or prevent cancer to a similar, identical, or greater extent than the parent TCR (e.g., depending on the applicable HLA molecule). In the context of the parent TCR, a functional portion may, for example, constitute about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95%, or more of the parent TCR.

[0065] A functional portion may comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, that are not found in the amino acid sequence of the parent TCR. Desirably, the additional amino acids do not interfere with the biological function of the functional portion, e.g., specifically binding to mutant p53 and / or having the ability to detect, treat or prevent cancer. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent TCR.

[0066] The polypeptide may comprise a functional portion of either or both of the α and β chains of the TCR of the present invention, for example, a functional portion comprising one or more of CDR1, CDR2, and CDR3 of the variable region(s) of the α and / or β chain of the TCR of the present invention. In an embodiment of the present invention, the polypeptide may comprise a functional portion comprising the amino acid sequence of: (1) all of SEQ ID NOs: 3 to 8; (2) all of SEQ ID NOs: 14 to 19; (3) all of SEQ ID NOs: 25 to 30; (4) all of SEQ ID NOs: 36 to 41; (5) all of SEQ ID NOs: 47 to 52; (6) all of SEQ ID NOs: 58 to 63; (7) all of SEQ ID NOs: 69 to 74; (8) all of SEQ ID NOs: 80 to 85; or (9) all of SEQ ID NOs: 131 to 136.

[0067] In an embodiment of the present invention, the polypeptide of the present invention may comprise, for example, a variable region of a TCR of the present invention comprising a combination of the above-mentioned CDR regions. In this regard, the polypeptide may be, for example: (1) both SEQ ID NOs:9 and 10; (2) both SEQ ID NOs:20 and 21; (3) both SEQ ID NOs:31 and 32; (4) both SEQ ID NOs:42 and 43; (5) both SEQ ID NOs:53 and 54; (6) both SEQ ID NOs:64 and 65; (7) both SEQ ID NOs:75 and 76; (8) both SEQ ID NOs:86 and 87; (9) both SEQ ID NOs:137 and 138; (10) both SEQ ID NOs:142 and 143; (11) both SEQ ID NOs:144 and 145; (12) both SEQ ID NOs:146 and 147; (13) both SEQ ID NOs:148 and 149; (14) both SEQ ID NOs:150 and 151; (15) both SEQ ID NOs:152 and 153; (16) both SEQ ID NOs:154 and 155; (17) both SEQ ID NOs:156 and 157; (18) both SEQ ID NOs:159 and 158; (19) both SEQ ID NOs:178 and 10; (20) both SEQ ID NOs:181 and 21; (21) both SEQ ID NOs:184 and 32; (22) both SEQ ID NOs:187 and 43; (23) both SEQ ID NOs:190 and 54; (24) both SEQ ID NOs:193 and 65; (25) both SEQ ID NOs:196 and 76; (26) both SEQ ID NOs:199 and 87; (27) both SEQ ID NOs:137 and 202; (28) both SEQ ID NOs:9 and 205; (29) both SEQ ID NOs:20 and 207 (30) both SEQ ID NOs:31 and 209; (31) both SEQ ID NOs:42 and 211; (32) both SEQ ID NOs:53 and 213; (33) both SEQ ID NOs:64 and 215; (34) both SEQ ID NOs:75 and 217; (35) both SEQ ID NOs:86 and 219; (36) both SEQ ID NOs:137 and 221; (37) both SEQ ID NOs:223 and 202; (38) both SEQ ID NOs:223 and 221; (39) both SEQ ID NOs:20 and 226; or (40) both SEQ ID NOs:181 and 226.

[0068] In an embodiment of the present invention, the polypeptide of the present invention may further comprise the constant region of the TCR of the present invention. In this regard, the polypeptide may comprise, for example, the amino acid sequence of (i) one of SEQ ID NOs: 91 to 93, or (ii) one of SEQ ID NOs: 93 and 91 and 92.

[0069] In an embodiment of the invention, the polypeptide of the invention may comprise the α and β chains of a TCR of the invention. In this regard, the polypeptide may comprise, for example: (1) both SEQ ID NOs: 11 and 12; (2) both SEQ ID NOs: 22 and 23; (3) both SEQ ID NOs: 33 and 34; (4) both SEQ ID NOs: 44 and 45; (5) both SEQ ID NOs: 55 and 56; (6) both SEQ ID NOs: 66 and 67; (7) both SEQ ID NOs: 77 and 78; (8) both SEQ ID NOs: 88 and 89; (9) both SEQ ID NOs: 139 and 140; (10) both SEQ ID NOs: 160 and 161; (11) both SEQ ID NOs: 120 and 122; (12) both SEQ ID NOs: 122 and 123; (13) both SEQ ID NOs: 124 and 125; (14) both SEQ ID NOs: 126 and 127; (15) both SEQ ID NOs: 128 and 129; (16) both SEQ ID NOs: 129 and 130; (17) both SEQ ID NOs: 131 and 132; (18) both SEQ ID NOs: 133 and 134; (19) both SEQ ID NOs: 135 and 136; (20) both SEQ ID NOs: 137 and 138; (21) both SEQ ID NOs: 138 and 139; (22) both SEQ ID NOs: 139 and 140; (23) both SEQ ID NOs: 139 and 140; (24) both SEQ ID NOs: (12) both SEQ ID NOs:164 and 165; (13) both SEQ ID NOs:166 and 167; (14) both SEQ ID NOs:168 and 169; (15) both SEQ ID NOs:170 and 171; (16) both SEQ ID NOs:172 and 173; (17) both SEQ ID NOs:174 and 175; (18) both SEQ ID NOs:176 and 177; (19) both SEQ ID NOs:179 and 12; (20) both SEQ ID NOs:182 and 23; (21 (22) both SEQ ID NOs:185 and 34; (22) both SEQ ID NOs:188 and 45; (23) both SEQ ID NOs:191 and 56; (24) both SEQ ID NOs:194 and 67; (25) both SEQ ID NOs:197 and 78; (26) both SEQ ID NOs:200 and 89; (27) both SEQ ID NOs:139 and 203; (28) both SEQ ID NOs:11 and 206; (29) both SEQ ID NOs:22 and 208; (30) both SEQ ID NOs:33 and 210; (31) SEQ ID NO:44 and 212; (32) both SEQ ID NOs:55 and 214; (33) both SEQ ID NOs:66 and 216; (34) both SEQ ID NOs:77 and 218; (35) both SEQ ID NOs:88 and 220; (36) both SEQ ID NOs:139 and 222; (37) both SEQ ID NOs:224 and 203; (38) both SEQ ID NOs:224 and 222; (39) both SEQ ID NOs:22 and 227; or (40) both SEQ ID NOs:182 and 227.

[0070] The present invention further provides a protein comprising at least one of the polypeptides described herein. By "protein" is meant a molecule comprising one or more polypeptide chains. In an embodiment, the protein of the present invention comprises: (1) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 3 to 5 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 6 to 8; (2) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 14 to 16 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 17 to 19; (3) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 25 to 27 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 28 to 30; (4) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 36 to 38 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 39 to 41; (5) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 47 to 49. and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 50 to 52; (6) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 58 to 60 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 61 to 63; (7) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 69 to 71 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 72 to 74; (8) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 80 to 82 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 83 to 85; or (9) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 131 to 133 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 134 to 136.

[0071] In an embodiment of the invention, the protein comprises: (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:10; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:20 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:21; (3) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:31 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:32; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:42 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:43; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:53 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:54; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:64 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:65; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:75 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:76; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:86 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:87; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 138; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 142 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 143; (11) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 144 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 145; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 146 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 147; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 148 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 149; (14) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 150 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 151; (15) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 152 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 153; (16) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 154 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 155;(17) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 156 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 157; (18) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 158 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 159; (19) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 178 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10; (20) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 181 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 21; (21) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 184 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32; (22) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 187 (23) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 190 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 54; (24) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 193 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 65; (25) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 196 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 76; (26) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 199 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 87; (27) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 137; (28) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 205; (29) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 20 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 207; (30) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 209; (31) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 211; (32) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 53 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 213; (33) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 215; (34) an amino acid sequence of SEQ ID NO: 75 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:217; (35) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:86 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:219; (36) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:137 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:221; (37) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:223 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:202; (38) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:223 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:221; (39) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:20 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:226; or (40) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:181 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:226.

[0072] In an embodiment of the invention, the protein comprises: (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:11 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:12; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:22 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:23; (3) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:33 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:34; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:44 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:45; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:55 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:56; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:66 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:67; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:77 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:78; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:88 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:89; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 140; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 161; (11) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 162 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 163; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 164 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 165; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 166 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 167; (14) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 169; (15) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 170 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 171; (16) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 172 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 173;(17) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 174 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 175; (18) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 177; (19) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 179; (20) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 182 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 23; (21) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 185 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34; (22) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 188 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45; (23) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 191 and (24) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 194 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67; (25) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 197 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 78; (26) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 200 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 89; (27) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 203. (28) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 11 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 206; (29) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 208; (30) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 210; (31) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 44 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 212. (32) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:55 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:214; (33) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:66 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:216; (34) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:77 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:218; (35) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:88 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:220;(36) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 222; (37) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 224 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 203; (38) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 224 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 222; (39) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 227; or (40) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 182 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 227.

[0073] The protein of the invention may be a TCR. Alternatively, the protein of the invention may be a fusion protein, when the first and / or second polypeptide chain(s) of the protein further comprises another amino acid sequence, for example an amino acid sequence encoding an immunoglobulin or a portion thereof. In this regard, the invention also provides a fusion protein comprising at least one of the polypeptides of the invention described herein, together with at least one other polypeptide. The other polypeptide may be present as a separate protein of a fusion protein, or as a polypeptide expressed in frame (in tandem) with one of the polypeptides of the invention described herein. The other polypeptide may encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to, an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.

[0074] A fusion protein may contain one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein may contain 1, 2, 3, 4, 5, or more copies of a polypeptide of the invention and / or another polypeptide. Suitable methods for making fusion proteins are known in the art and can include, for example, recombinant methods. Some examples include:

[0075] In some embodiments of the invention, the TCRs, polypeptides, and proteins of the invention may be expressed as a single protein comprising a linker peptide linking the α and β chains. In this regard, the TCRs, polypeptides, and proteins of the invention may further comprise a linker peptide. The linker peptide may advantageously facilitate expression of the recombinant TCRs, polypeptides, and / or proteins in a host cell. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may comprise the amino acid sequence of SEQ ID NO: 94. Once the construct comprising the linker peptide is expressed by the host cell, the linker peptide may be cleaved, resulting in separated α and β chains. In embodiments of the invention, the TCRs, polypeptides, or proteins may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains.

[0076] In some embodiments, a TCR, polypeptide or protein disclosed herein comprises an α chain and / or a β chain as disclosed herein, including a signal peptide. In some embodiments, the sequence of the signal peptide of any of the α chain and / or β chain disclosed herein comprises an alanine or histidine residue substituted for the wild-type residue at position 2.

[0077] In some embodiments, the TCRs, polypeptides or proteins disclosed herein comprise the mature forms of the α and / or β chains as disclosed herein, lacking the signal peptide.

[0078] The protein of the invention may be a recombinant antibody or antigen-binding portion thereof, comprising at least one of the polypeptides of the invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the invention and an antibody polypeptide chain or antigen-binding portion thereof. The antibody polypeptide or antigen-binding portion thereof may be an antibody heavy chain, light chain, variable or constant region of the heavy or light chain, a single chain variable region (scFv), or an Fc, Fab, or F(ab) polypeptide. 2 The antibody polypeptide chains or antigen-binding portions thereof may be present as separate polypeptides in a recombinant antibody. Alternatively, the antibody polypeptide chains or antigen-binding portions thereof may be present as polypeptides expressed in frame (tandem) with a polypeptide of the invention. The antibody polypeptide or antigen-binding portion thereof may be the polypeptide of any antibody or any antibody fragment, including any of the antibodies and antibody fragments described herein.

[0079] The TCRs, polypeptides, and proteins of the invention may be of any length, i.e., contain any number of amino acids, so long as they retain their biological activity, e.g., the ability to specifically bind to mutant p53, detect cancer in a mammal, or treat or prevent cancer in a mammal. For example, the polypeptides may be in the range of about 50 to about 5000 amino acids in length, e.g., 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 invention also include oligopeptides.

[0080] The TCRs, polypeptides and proteins of the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans. Examples of suitable glyceryl esters include cis-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0081] The TCRs, polypeptides and proteins of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or multimerized, or conjugated.

[0082] The TCRs, polypeptides, and / or proteins of the invention can be obtained by methods known in the art, such as de novo synthesis. Polypeptides and proteins can also be produced recombinantly using standard recombinant techniques and the nucleic acids described herein. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual ,4 thed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Alternatively, companies such as, for example, Synpep (Dublin, CA), Peptide Technologies Corp. (Gaithersburg, MD), and Multiple Peptide Systems (San Diego, CA) can commercially synthesize the TCRs, polypeptides, and / or proteins described herein. In this regard, the TCRs, polypeptides, and proteins of the invention may be synthetic, recombinant, isolated, and / or purified.

[0083] An embodiment of the present invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. As used herein, "nucleic acid" includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules," and generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. In an embodiment, a nucleic acid comprises a complementary DNA (cDNA). It is generally preferred that a nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, it may be preferred that a nucleic acid contains one or more insertions, deletions, inversions, and / or substitutions, as discussed herein.

[0084] Preferably, the nucleic acid of the present invention is recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by linking a natural or synthetic nucleic acid segment to a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule obtained by replicating what is described in (i) above. For purposes herein, the replication may be in vitro or in vivo.

[0085] 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, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylquefine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted adenines, and the like. Examples of nucleic acids that may be used include, but are not limited to, uracil, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of the present invention can be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX).

[0086] In an embodiment 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 a particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence increases the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but that can be translated by a more readily available tRNA in the cell, thereby increasing translation efficiency. Optimization of a nucleotide sequence may also reduce secondary structures in the mRNA that interfere with translation, thereby increasing translation efficiency.

[0087] The present invention also provides a nucleic acid comprising a nucleotide sequence that is complementary to the nucleotide sequence of any of the nucleic acids described herein, or that hybridizes under stringent conditions to the nucleotide sequence of any of the nucleic acids described herein.

[0088] Nucleotide sequences that hybridize under stringent conditions preferably hybridize under high stringency conditions. By "high stringency conditions" is meant that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably greater than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides having exact complementary sequences or those containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3-10 bases) that match the nucleotide sequence. Such small regions of complementarity melt more easily than full-length complements of 14-17 or more bases, making them easily distinguishable by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by, for example, about 0.02-0.1 M NaCl or equivalent, and temperatures of about 50-70° C. Such high stringency conditions are suitable for the detection of nucleotides that are highly complementary to the target sequence. It tolerates little, if any, mismatch between the primer sequence and the template or target strand and is particularly suitable for detecting expression of any of the TCRs of the present invention. It will generally be appreciated that conditions can be made more stringent by adding increasing amounts of formamide.

[0089] The invention also provides nucleic acids comprising a nucleotide sequence that is at least about 70% or more identical to any of the nucleic acids described herein, e.g., about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein. In this regard, the nucleic acid may consist essentially of any of the nucleotide sequences described herein.

[0090] 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 embodiment of the invention, the recombinant expression vector comprises a nucleotide sequence encoding an α chain, a β chain, and a linker peptide.

[0091] For purposes herein, the term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and causes a cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with a host cell under conditions sufficient to cause expression of the mRNA, protein, polypeptide, or peptide in the cell. The vector of the present invention is not naturally occurring in its entirety. However, portions of the vector may be naturally occurring. The recombinant expression vector of the present invention may contain any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring internucleotide bonds, non-naturally occurring internucleotide bonds, or both types of bonds. Preferably, the non-naturally occurring or modified nucleotides or internucleotide bonds do not interfere with the transcription or replication of the vector.

[0092] The recombinant expression vector of the present invention may be any suitable recombinant expression vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and propagation, or for expression, or both, such as plasmids and viruses. The vector may be selected from the group consisting of the transposon / transposase series, pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors such as λGT10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). Preferably, the recombinant expression vector is a transposon vector or a viral vector, such as a retroviral vector.

[0093] The recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques, for example as described in Green and Sambrook et al., supra. Linear expression vector constructs can be prepared that contain replication systems that function in prokaryotic or eukaryotic host cells, for example, replication systems derived from ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0094] Desirably, the recombinant expression vector includes control sequences, e.g., transcription and translation initiation and termination codons that are specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced, as appropriate, and considering whether the vector is DNA or RNA based.

[0095] The recombinant expression vector may contain one or more marker genes to allow for the selection of transformed or transfected host cells. Marker genes include biocide resistance, resistance to e.g., antibiotics, heavy metals, etc., complementation in auxotrophic hosts to confer prototrophy, etc. Suitable marker genes for the expression vectors of the present invention include, for example, the neomycin / G418 resistance gene, the hygromycin resistance gene, the histidinol resistance gene, the tetracycline resistance gene, and the ampicillin resistance gene.

[0096] The recombinant expression vector may include a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or to a nucleotide sequence that is complementary or hybridizes to a nucleotide sequence encoding a TCR, polypeptide, or protein. For example, the selection of strong, weak, inducible, tissue-specific, and developmental stage-specific promoters is within the skill of one of ordinary skill in the art. Similarly, the combination of a nucleotide sequence and a promoter is within the skill of one of ordinary skill in the art. The promoter may be a non-viral promoter, such as the human elongation factor-1 alpha promoter, or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and the promoter found in the long terminal repeat of murine stem cell virus.

[0097] The recombinant expression vectors of the invention can be designed for transient expression, stable expression, or both. Additionally, the recombinant expression vectors can be made for constitutive or inducible expression.

[0098] Additionally, the recombinant expression vector may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the death of a cell expressing the suicide gene. A suicide gene may be a gene that confers sensitivity to an agent, such as a drug, on the cell in which it is expressed, and causes the cell to die when contacted or exposed to the agent. Suicide genes are known in the art and include, for example, herpes simplex virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0099] Another embodiment of the present invention further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that may contain a recombinant expression vector of the present invention. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or a protozoan. The host cell may be a cultured cell or a primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in a suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Preferably, the host cell is an animal cell. Most preferably, the host cell is a human cell. The host cell may be of any cell type, derived from any type of tissue, and at any stage of development, but preferably, the host cell is a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0100] For purposes herein, a T cell may be any T cell, e.g., a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells may also be enriched or purified. Preferably, the T cells are human T cells. The T cells may be any type of T cell, may be at any stage of development, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0101] Also provided by the present invention is a population of cells comprising at least one host cell as described herein. The population of cells may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any of the recombinant expression vectors, or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogenous population comprising primarily host cells comprising (e.g., consisting essentially of) a recombinant expression vector. The population may also be a clonal population of cells, where all cells of the population are clones of one host cell comprising a recombinant expression vector, such that all cells of the population comprise a recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising a recombinant expression vector as described herein.

[0102] In embodiments of the present invention, the number of cells in a population can be rapidly expanded. Expansion of T cell numbers can be achieved by, for example, expanding the number of T cells using methods described in U.S. Patent Nos. 8,034,334, 8,383,099, U.S. Patent Application Publication No. 2012 / 0244133, Dudley et al., J. Immunother., 26:332-42 (2003), and Riddell et al. Expansion of T cell numbers can be accomplished by any of a number of methods known in the art, such as those described in (E. et al., J. Immunol. Methods, 128:189-201 (1990). In an embodiment, expansion of T cell numbers is carried out by culturing the T cells with OKT3 antibody, IL-2, and feeder PBMCs (e.g., irradiated allogeneic PBMCs).

[0103] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the present invention may be isolated and / or purified. The term "isolated" as used herein means removed from its natural environment. The term "purified" as used herein means increased purity, and "purity" is a relative term and is not necessarily to be construed as absolute purity. For example, the purity may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or may be about 100%.

[0104] The TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, and host cells (including populations thereof) of the invention (all of which are hereinafter collectively referred to as the "TCR material of the invention") may be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention relates to The present invention provides a pharmaceutical composition comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described in and a pharma- ceutical acceptable carrier. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention may contain more than one TCR material of the invention, e.g., polypeptides and nucleic acids, or two or more different TCRs. Alternatively, the pharmaceutical composition may contain the TCR material of the invention in combination with another pharma- ceutical active agent(s) or drug(s), e.g., chemotherapeutic agents such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0105] Preferably, the carrier is a pharma- ceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the present invention under consideration. Methods for preparing administrable compositions are known or apparent to those skilled in the art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 22 nd Ed., Pharmaceutical Press (2012). Preferably, a pharma- ceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0106] The choice of carrier is determined in part by the specific TCR material of the present invention, as well as by the specific method used to administer the TCR material of the present invention.Therefore, there are various suitable formulations of the pharmaceutical composition of the present invention.Suitable formulations may include those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral, or intraperitoneal administration.More than one route may be used to administer the TCR material of the present invention, and in certain instances, a particular route may provide a more immediate and effective response than another route.

[0107] Preferably, the TCR material of the invention is administered, for example, by injection intravenously. When the TCR material of the invention is a host cell expressing a TCR of the invention, the pharma- ceutically acceptable carrier for the cells for injection can include any isotonic carrier, such as normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5% dextrose in water, or lactated Ringer's solution. In an embodiment, the pharma- ceutically acceptable carrier is supplemented with human serum albumin.

[0108] For purposes of the present invention, the amount or dose of the TCR material of the invention administered (e.g., number of cells if the TCR material of the invention is one or more cells) should be sufficient to provide an effect, e.g., a therapeutic or prophylactic response, in the subject or animal over a suitable time frame. For example, a dose of the TCR material of the invention should be sufficient to bind a cancer antigen (e.g., mutant p53) or detect, treat, or prevent cancer for a period of about 2 hours or more from the time of administration, e.g., 12-24 hours or more. In certain embodiments, the period may be even longer. The dose will be determined by the efficacy of the particular TCR material of the invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) to be treated.

[0109] Many assays for determining the dose to be administered are known in the art. For purposes of the present invention, a starting dose to be administered to a mammal can be determined using an assay that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a given dose of a TCR, polypeptide, or protein of the present invention when administered to the mammal, between a set of mammals that have each received different doses of T cells. The degree to which target cells are lysed or IFN-γ is secreted by such T cells when administered a particular dose can be determined using an assay that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by such T cells when administered to a mammal, between a set of mammals that have each received different doses of T cells. The degree to which it is secreted can be assayed by methods known in the art.

[0110] The dose of the TCR material of the invention will also be determined by the existence, nature, and extent of any adverse side effects that may accompany the administration of a particular TCR material of the invention. Typically, the attending physician will determine the dosage of the TCR material of the invention for treating each individual patient, taking into account a variety of factors, such as age, weight, general health, diet, sex, the TCR material of the invention to be administered, the route of administration, and the severity of the cancer being treated. In embodiments where the TCR material of the invention is a population of cells, the number of cells administered per injection may be, for example, about 1×10 6 ~Approx. 1×10 12 The number of cells may vary from 1×10 to 10×10 or more. 6 Subcellular amounts may also be administered.

[0111] Those skilled in the art will readily appreciate that the TCR material of the present invention may be modified in any number of ways, resulting in increased therapeutic or prophylactic efficacy of the TCR material of the present invention through such modifications. For example, the TCR material of the present invention may be conjugated to a chemotherapeutic agent, either directly or indirectly via cross-linking. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art will appreciate that sites of the TCR material of the present invention that are not essential to the function of the TCR material of the present invention are ideal sites for binding cross-linking and / or chemotherapeutic agents, as long as the cross-linking and / or chemotherapeutic agent does not interfere with the function of the TCR material of the present invention, i.e., its ability to bind mutant p53 or detect, treat, or prevent cancer, upon binding to the TCR material of the present invention.

[0112] It is contemplated that the pharmaceutical compositions, TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells of the invention can be used in methods of treating or preventing cancer. Without being bound to a particular theory, it is believed that the TCRs of the invention specifically bind to mutant p53, such that the TCRs (or related polypeptides or proteins of the invention), when expressed by a cell, can mediate an immune response against target cells expressing mutant p53. In this regard, embodiments of the invention provide a method of treating or preventing cancer in a mammal, comprising administering to the mammal any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, in an amount effective to treat or prevent cancer in the mammal.

[0113] Embodiments of the invention provide any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0114] The terms "treat" and "prevent," and words derived therefrom, as used herein do not necessarily mean 100% or complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art would recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any amount or level of treatment or prevention of cancer in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, the treatment or prevention can include promoting tumor regression. Also, for purposes herein, "prevention" refers to delaying the onset of cancer or its symptoms or conditions. Alternatively, or in addition, "prophylaxis" can include preventing or delaying the recurrence of cancer or a symptom or condition thereof.

[0115] Also provided by embodiments of the invention is a method of detecting the presence of cancer in a mammal, the method comprising: (i) contacting any of the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions of the invention described herein with a sample comprising one or more cells from a mammal, thereby forming a complex, and detecting the complex, wherein detection of the complex indicates the presence of cancer in the mammal.

[0116] For the present methods of detecting cancer in a mammal, the cellular sample may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, such as a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

[0117] For purposes of the detection method of the present invention, contacting may be performed in vitro or in vivo in a mammal, preferably in vitro.

[0118] Detection of the complex can also be accomplished through any number of methods known in the art. For example, the TCRs, polypeptides, proteins, nucleic acids, recombinant expression vectors, host cells, or populations of cells of the invention described herein may be labeled with a detectable label, such as a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase), and an elemental particle (e.g., gold particle), etc.

[0119] For purposes of the methods of the invention in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0120] In the context of the methods of the present invention, cancer can be, for example, acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors. The cancer may be any cancer, including any of the following: glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharynx cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. In a preferred embodiment, the cancer is a cancer that expresses a mutant p53. The cancer may express a p53 with a mutation at one or both of positions 175 and 220 as defined by SEQ ID NO: 1. The cancer may express a p53 with one or both of the following human p53 mutations: R175H and Y220C. Preferably, the cancer is an epithelial cancer, or cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

[0121] The mammal referred to in the methods of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals, such as mice and hamsters, and lagomorph mammals, such as rabbits. Preferably, the mammal is a mammal of the carnivora order, which includes cats and dogs. Preferably, the mammal is a mammal of the even-toed ungulate order, which includes cats and pigs, or the odd-toed ungulate order, which includes horses. More preferably, the mammal is a mammal of the order Primates, Ceboids or Simoids (monkeys), or suborder Anthropoids (humans and apes). A particularly preferred mammal is a human. EXAMPLES

[0122] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0123] In the experiments described in Examples 1-7, the following materials and methods were used.

[0124] Subjects and Samples Leukapheresis products and tumor samples were obtained from individuals with metastatic epithelial cancer enrolled in National Institutes of Health protocol NCT01174121, approved by the National Cancer Institute (NCI) Institutional Review Board (IRB). Patients were selected based on the availability of pretreatment leukapheresis and TIL screening results and had undergone prior treatment (surgery, chemotherapy, radiotherapy) according to standard of care (Malekzadeh et al., J. Clin. Invest. 129(3): 1109-14(2019); Deniger et al., Clin. Cancer Res., 24(22):5562-73(2018)). PBLs were isolated from leukapheresis using Ficoll-Hypaque, and cells were cryopreserved for future use. All patients studied had TP53 mutations confirmed by whole-exome sequencing according to clinical protocols as previously described (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019)).

[0125] Antibodies and FACS Fluorescently labeled antibody flow cytometry is detailed in Table 1. Flow cytometry analysis was performed on a FACS CANTO II system (BD Biosciences, San Jose, CA) with analysis by FLOWJO software (TreeStar, Ashland, OR). Total cells were gated on lymphocytes and live cells by excluding cells stained with propidium iodide (PI). Cells were sorted for IVS and 4-1BB / OX40 on a FACS ARIA II cell sorter (BD Biosciences). 4-1BB + and / or OX40 + The cells were then subjected to + CD4 + CD8 - (CD4) and CD3 + CD4 - CD8 + The co-cultures were sorted by SH800S cell sorter (Sony Biotechnology) for single-cell PCR to identify TCR genes.

[0126] [Table 1]

[0127] TP53 "hot spot" mutation screening reagent For TIL screening, TMG constructs of WT (TMG-WT-TP53) and mutant TP53 (TMG-MUT-TP53) were generated as previously described (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019)). Briefly, each TP53 "hotspot" mutation (R175H, Y220C, G245S, G245D, R248L, R248Q, R248W, R249S, R273C, R273H, R273L, and R282W) was constructed into a minigene with the mutant codon in the middle and 12 normal codons upstream and downstream, and the minigene was ligated into TMG. The analogous sequence corresponding to the WT sequence was also derived. TMG was synthesized as DNA, cloned in frame with the LAMP signal sequence and DC-LAMP localization sequence, and then transcribed in vitro into mRNA using the mMESSAGE mMACHINE T7 UltraKit according to the manufacturer's instructions (Thermo-Fisher; Waltham, MA). R175H , p53 R248W , and p53 Y220C The peptides were synthesized and purified to >95% by high performance liquid chromatography (Genscript; Piscataway, NJ). All peptides were reconstituted in DMSO.

[0128] antigen presenting cells Monocyte-derived immature dendritic cells (DCs) were generated by the adherence method (Tran et al., Science, 350(6266):1387-90(2015)). Briefly, PBLs were plated in AIM-V medium (Life Technologies, Waltham, MA) containing DNase (Genentech Inc., San Francisco, CA) and incubated at 37°C for 1.5-2 hours. Non-adherent cells were removed and used fresh or cryopreserved. Adherent cells were washed with AIM-V, incubated at 37°C for 1 hour, and the medium was replaced with DC medium (5% human serum, 1% CO). The medium was then switched to RPMI-1640, 2 mM L-glutamine, containing 0.00 U / mL penicillin, 100 μg / mL streptomycin, amphotericin B, 800 IU / mL granulocyte-macrophage colony-stimulating factor (GM-CSF), and 200 U / mL interleukin-4 (IL-4) (Peprotech, Rocky Hill, NJ). Cells were fed every 2-3 days and harvested on days 5-6.

[0129] IVS, mutant TP53 co-culture, 4-1BB / OX40 enrichment and REP To perform antigen-experience selection, pretreated PBLs were treated as previously described (Cafri et al., Nat. Commun., 10(1):449(2019)). Cryopreserved apheresis samples were thawed, washed, and diluted at 5–10x10 in AIM-V medium containing DNase. 6 Set to 1.75-2x10 cells / ml 8 Viable cells were plated in T175 flasks (Corning Inc., Corning, NY) and incubated at 37°C and 5% CO 2 After 90 min, non-adherent monocyte-depleted PBLs were harvested, centrifuged, and cultured in 50 / 50 medium (AIM-V medium, RPMI-1640 medium (Lonza, Walkersville, MD), 5% human AB serum, 100 U / mL penicillin and 100 μg / mL streptomycin (Life Technologies), 2 mM L-glutamine (Life Technologies), 10 μg / mL gentamicin (Quality Biological Inc., Gaithersburg, MD), 12.5 mM HEPES (Life Technologies)) at 37°C and 5% CO. 2 The adherent monocytes were differentiated into immature DCs as described above. After leaving the non-adherent cells overnight, the cells were harvested and cultured at 1–2 × 10 8Cells were resuspended in 50 μl of staining buffer (PBS, 0.5% BSA, 2 mM EDTA) containing CD3, CD8, CD4, CD62L, and CD45RO antibodies. Cells were incubated at 4°C for 30 min and washed twice before acquisition. To determine sorted populations, live cells (propidium iodide negative), single cells, CD3 + T cells, followed by antigen-experienced cells (CD62L - CD45RO + , CD62L + CD45RO + , CD62L - CD45RO - ) and further gated with CD4 + or CD8 + CD8 + and CD4 + Antigen-experienced memory T cells were separately sorted, collected, counted, and resuspended in 50 / 50 medium containing interleukin-21 (IL-21) at a concentration of 60 ng / mL. Autologous DCs were electroporated with TMG-MUT-TP53 18–24 h before the day of FACS sorting or pulsed with patient-specific mutant p53-LP for 2–4 h on that day. Target cells (DCs) were washed twice with 50 / 50 medium and resuspended in 50 / 50 medium without cytokines. After addition of DCs, in vitro stimulation (IVS) was performed in co-cultures at a ratio of 1:3–1:6 (DC:T cells) with IL-21 at a final concentration of 30 ng / mL. After 14 days of expansion and three doses of IL-21 and interleukin-2 (IL-2; aldesleukin), autologous DCs were again electroporated or pulsed with TMG-MUT-TP53 or mutant long peptide (LP), respectively, and IVS cultures were incubated at 37°C and 5% CO as previously described (Cafri et al., Nat. Commun., 10(1):449 (2019)). 2DCs were co-cultured with DC electroporated with irrelevant TMG or with DC pulsed with DMSO for 18-24 hours. In parallel, for negative control during 4-1BB / OX40 enrichment sorting, IVS cultures were co-cultured with DCs electroporated with irrelevant TMG or with DCs pulsed with DMSO. After co-culture, cells were harvested and resuspended in 50 μl staining buffer (PBS, 0.5% BSA, 2 mM EDTA) containing CD3, CD4, CD8, 4-1BB and OX40 antibodies, incubated for 30 min at 4°C and washed twice before acquisition. Sorted 4-1BB + / OX40 + Enriched T cells were expanded by REP using irradiated PBL feeders, 30 ng / mL OKT3 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany), and 3,000 IU / mL IL-2 in 50 / 50 medium. Rapid expansion (REP) cultures were fed three times and tested for reactivity on day 14 or cryopreserved.

[0130] co-culture Screening of IVS and enriched T cells was achieved by the same strategy used to screen TIL fragments (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14 (2019)). Briefly, autologous DCs were cultured with TMG (10 5 cells / well) and left overnight or pulsed with peptide or DMSO for 2-4 hours (8x10 4 Target cells were washed twice, resuspended in 50 / 50 medium, and plated at 2x10 cells / well in Enzyme-Linked ImmunoSpot (ELISPOT) plates for interferon-γ (IFNγ) (EMD Millipore, Burlington, MA). 4Tumor cells were co-cultured with T cells. Phorbol 12-myristate 13-acetate (PMA) and ionomycin (Thermo Fisher) were used as positive controls, and medium alone was the negative control. Co-cultured cells were removed, stained, and analyzed by flow cytometry as described above, while ELISPOT plates were processed according to the manufacturer's instructions. Tumor cell lines were grown from cryopreserved stocks for at least 1 week before co-culture. Their generation is described elsewhere (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019)). Tumor cells were co-cultured with T cells (a total of 2x10 5 Cells) at a 1:1 ratio overnight. Co-culture supernatants were collected and IFNγ secretion was assessed by enzyme-linked immunosorbent assay (ELISA; ThermoFisher), and the co-cultured cells were analyzed by flow cytometry.

[0131] Shortest peptide assay and HLA restriction mapping The shortest peptides were identified and HLA-restricted using a similar method previously described (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019)). Briefly, a peptide binding affinity algorithm (v3.4) was used (Lundegaard et al., Bioinformatics, 24(11):1397-8(2008)) to predict neoepitopes for HLA class I alleles. Candidates 9-11 amino acids were co-cultured as described above. CD4 + To investigate the shortest neoantigens, 15 amino acid peptides overlapping by 14 amino acids were co-cultured as described above. To determine HLA restriction, COS7 tumor cells were cultured at 2.5x10 in RPMI-1640, 2mM L-glutamine and 10% fetal bovine serum in flat-bottom 96-well plates. 4Cells / well were plated and incubated overnight at 37°C. DNA plasmids (pcDNA3.1 backbone) with patient-specific individual HLA class I alleles (300ng / well) or both HLA class II α and β chains (150ng / well each) were transfected with LIPOFECTAMINE2000 transfection reagent according to the manufacturer's instructions (ThermoFisher). When TMG was co-transfected with HLA, the concentration of HLA was reduced to 150ng / well for class I and 100ng / well for class II. The WT TMG used for these experiments was TMG-MUT-TP53 reverted to WT only at the desired position, e.g., R175H. After 24 hours of incubation, the transfection medium was removed and peptide or DMSO was pulsed in 50 / 50 medium for 2-4 hours, wells were washed twice with 50 / 50 medium, and 10 5 T cells were incubated overnight. Co-culture supernatants were analyzed for IFNγ secretion by ELISA, and cells were stained for upregulation of 4-1BB and analyzed by flow cytometry.

[0132] TCRB sequencing TCRB survey sequencing was performed from genomic DNA by Adaptic Biotechnologies (Seattle, WA). A minimum of 5x10 DNA was used for sequencing. 4 The analysis of productive TCR rearrangements was performed using IM This was performed using the MUNOSEQ ANALYZER 3.0 tool (Adaptive Biotechnologies).

[0133] TCR Identification and Reconstitution Similar to previous studies (Pasetto et al., Cancer Immunol. Res., doi 10.1158 / 2326-6066.CIR-16-0001 (2016); Deniger et al., Clin. Cancer Res., 23(2):351-62(2017)), co-cultures of T cells and DCs expressing p53 neoantigens were sorted into single wells and subsequently TCRs were identified by single-cell reverse transcriptase polymerase chain reaction (RT-PCR) of the TCR gene. PCR products were maintained separately for TCRα and TCRβ and analyzed by Sanger sequencing. These partial TCR sequences were analyzed by the IMGT / V-Quest (imgt.org / IMGT) and IGBLAST (ncbi.nlm.nih.gov / igblast) websites, which identify the exact CDR3 and J or D / J regions as well as the most likely TRAV and TRBV families. As done in other studies, human full-length variable sequences were fused to mouse constant chains (Deniger et al., Clin. Cancer Res., 23(2):351-62(2017); Cohen et al., J. Clin. Invest., 125(10):3981-91(2015)). The humanized TCRα and TCRβ genes were ligated with the RAKR-SGSG (SEQ ID NO: 128) and P2A ribosome slip sequences, resulting in stoichiometric expression of the TCR in a single cistron. This sequence was synthesized for the generation of transient retroviral supernatants and cloned into the MSGV1 vector.

[0134] TCR transduction PBL donors were cultured in 50 / 50 medium supplemented with 50 ng / ml of soluble OKT3 and 300 IU / ml of IL-2 at 3x10 6The cells were adjusted to 10 cells / ml and activated on reduced adhesion plates for 2 days before retroviral transduction. Mutation-specific TCR-encoding pMSGV1 plasmid (1.5 μg / well) and envelope-encoding plasmid RD114 (0.75 μg / well) were transfected into 10 wells of a 6-well poly-D-lysine-coated plate using LIPOFECTAMINE 2000 transfection reagent (Life Technologies). 6 The retroviral supernatant was collected 2 days after transfection, diluted 1:1 with DMEM medium, and centrifuged twice at 2000xg and 32°C for 2 hours onto non-tissue culture treated 6-well plates coated with RETRONECTIN reagent (10μg / well, Takara, Rockville, MD). The supernatant was aspirated and 2x10 6 Stimulator T cells were cultured at 5x10 in 50 / 50 medium containing 300IU / mL IL-2. 5 T cells were centrifuged at 300xg for 10 min onto plates coated with RETRONECTIN reagent. Media was replaced with 300IU / mL IL-2 3-4 days later, and transduced cells were assayed 10-14 days after transduction.

[0135] Example 1 This example demonstrates IVS of antigen-experienced peripheral blood T cells carrying a TP53 mutation.

[0136] Antigen-experienced T cells from PBL were evaluated in seven colon cancer patients, one rectal cancer patient, and one ovarian cancer patient with TP53-mutated tumors (Table 2). Patient 4217 (p53 R175H ), 4213(p53 R248Q ), 4257(p53 R248W ) and 4254(p53 R273H ) did not demonstrate a TIL response to the p53 neoantigen. In contrast, TILs were not observed in patient 4141 (p53 R175H ), 4285(p53 R175H ), 4149(p53 Y220C ), 4266(p53R248W ) and 4273(p53 R2 48W ) reacted to autologous TP53 mutations (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019); Deniger et al., Clin. Cancer Res., 24(22):5562-73(2018)). Using cryopreserved apheresis (prior to any ACT), antigen-experienced CD4 + or CD8 + T cells were selected (Figure 1A, left). Selected CD4 + or CD8 + T cells were in vitro stimulated with DCs expressing mutant TP53-TMG mRNA (TP53-TMG-IVS) or pulsed with patient-specific mutant p53-LP (p53-LP-IVS). After 12 days of culture in the presence of IL-21 and IL-2, in vitro stimulated CD4 + and CD8 + Memory cells were co-cultured with DCs electroporated with mutant TP53-TMG mRNA for TP53-TMG-IVS or with patient-specific mutant p53-LP for p53-LP-IVS, and selected the next day based on the expression of T cell activation markers 4-1BB and / or OX40 (Fig. 1A, center). Cell yields after TP53-TMG-IVS and p53-LP-IVS were CD4 + and CD8 + T cells were equivalent for both, 0.3x10 7 ~15.6x10 7 Starting with 0.9x10 T cells, 7 ~18.3x10 7 The range of CD4 + 4-1BB + / OX40 + and CD8 + 4-1BB + / OX40 + A fraction of T cells was selected from all populations for experimental completeness and equivalence, with 2x10 2 ~1.7x10 5Cells, parental CD4 or CD8 gates ranged from 0.1% to 6.9% (Table 3). Sorted T cells underwent REP and were analyzed after 14 days of rapid expansion. The final cell yield was 7x10 6 ~4.2x10 8 T cells and may have been influenced by the number of input cells to the REP (Table 3 ).

[0137] [Table 2]

[0138] [Table 3]

[0139] Example 2 This example demonstrates that TP53 mutation-reactive T cells were present in the PBL of patients with intratumoral TIL responses against the p53 neoantigen.

[0140] Analytical screening was performed on the cultures of Example 1 after IVS, 4-1BB / OX40 enrichment and REP, where reactivity was assessed by flow cytometry using the cell surface marker 4- The tumor response was assessed by upregulation of 1BB and by IFNγ secretion using an ELISPOT assay (Figure 1A, right). Peripheral blood T cells did not respond to the p53 neoantigen in patients 4213, 4217, 4254, and 4257, supporting the results of TIL screening (Table 2). These patients may not have had an immunogenic combination of HLA and p53 neoepitopes. In contrast, five patients (4141 and 4285: p53) showed intratumoral T cell responses to mutant TP53 by TILs. R175H , 4149:p53 Y220C , 4266 and 4273:p53 R248WTP53 mutation-specific T cells were identified among antigen-experienced T cells from PBLs of 163 mice (Figures 1B-1C and Table 2) (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019)). TP53-TMG-IVS yielded p53 neoantigen-specific T cells in 4141-CD8, 4285-CD4, 4149-CD4 and 4266-CD8 cultures (Figure 1B), whereas p53-LP-IVS yielded p53 neoantigen-specific T cells in 4285-CD4 and 4273-CD4 cultures (Figure 1C). The specificity of the response to mutant TP53 (filled shapes in Figures 1B-1C) was illustrated by the lack of response to the WT counterpart (open shapes in Figures 1B-1C). The highest frequency of TP53 mutation-reactive cells was observed in p53 R175H In 4141-CD8 TP53-TMG-IVS cultures, the percentage of CD8 T cells expressing the mutant TMG-MUT-TP53 and p53 R175H Responses to LP were not observed in mice receiving WT TMG-MUT-TP53 (irrelevant TMG), DMSO (peptide vehicle), or WT p53 R175 The positive results of the IFNγ secretion screen were exemplified by the 4285-CD4 TP53-TMG-IVS cultures, but not by LP (Figure 1E). R175H LP has the amino acid sequence YKQSQHMTEVVR H CPHHERCSDSDG (SEQ ID NO: 111). WT p53 R175 LP has the amino acid sequence YKQSQHMTEVVR R CPHHERCSDSDG (SEQ ID NO: 110). Selected cultures were deemed responsive based on upregulation of 4-1BB and / or IFNγ secretion and were studied further (Tables 4A-4B). 4285-CD4 TP53-TMG-IVS cultures upregulated the cognate p53 at peptide concentrations up to 10 ng / mL. R175H We demonstrated specific recognition of LP by IVS and 4-1BB / OX40 enrichment (Figure 1F). Thus, we identified highly specific CD8 + and CD4 + T cells could be identified.

[0141] [Table 4-1]

[0142] [Table 4-2]

[0143] Example 3 This example demonstrates that TCRB tracing demonstrated enrichment of p53 neoantigen-reactive T cells from PBL.

[0144] We wished to further characterize the TCR diversity for each patient's TP53 mutant-reactive T cell population and determine whether IVS and 4-1BB / OX40 enrichment altered the T cell repertoire. To accomplish this, we performed TCR deep sequencing (Pasetto et al., Cancer Immunol. Res., 4(9):734-43 (2016)) to measure productive T cell clonotype frequency based on unique CDR3B sequences and overall sample clonality. This is a normalized measure of population diversity that approaches 1 with more oligoclonal samples (Boyd et al., Sci. Transl. Med., 1(12):12ra23) (2009); Howie et al., Sci. Transl. Med., 7(301):301ra131 (2015)). TCRB clonality was significantly increased in TP53-TMG-IVS and p53-LP-IVS cultures generated from all patient PBL samples compared to pre-IVS PBL (Figure 2A). Similarly, the most frequent unique TCRB clonotypes in each TP53-TMG-IVS and p53-LP-IVS sample were more frequent than in pre-IVS PBL (Figure 2B). Ranking of p53 neoantigen-reactive TCRB sequences ranged from 1 to 167 in the final p53-LP-IVS or TP53-TMG-IVS cultures, but were either not detectable or ranked 5,020 in primary PBL (Table 5). Increased clonality or maximum TCRB clonotype frequency was not restricted to cultures with T cell responses to p53 neoantigen (Figures 2A-2B; asterisks indicate positive cultures). This suggested that there was a skewed T cell repertoire via IVS and 4-1BB / OX40 enrichment, but assessment of peak frequency and clonality was insufficient to predict the response of cultures to mutant TP53.

[0145] [Table 5]

[0146] Example 4 This example demonstrates the isolation of p53 neoantigen-reactive TCRs and tracking by TCRB sequencing.

[0147] We then identified TCRs from the p53 neoantigen-reactive IVS population for potential therapeutic and research applications, and to track TCR clonotypes over the culture period to assess the degree of TP53 mutation-reactive T cell enrichment. TCRs were identified as previously described in our previous study (Pasetto et al. l., Cancer Immunol. Res., 4(9):734-43 (2016)), as well as co-culture of reactive IVS cultures with cognate p53 neoantigens (TMG or LP), 4-1BB + Identification was performed by sorting T cells and undergoing single-cell RT-PCR of the TCR alpha and beta genes. The TCR pairs were reconstituted, cloned into retroviral vectors, and transduced into donor PBLs. The second amino acid residue was changed to alanine (A) to have a stronger Kozak sequence for highly efficient translation. The TCR alpha chain constant region was replaced with a cysteine-substituted, LVL-modified mouse alpha chain constant region. The TCR beta chain constant region was replaced with a cysteine-substituted mouse beta chain constant region.

[0148] p53 R175H (Patients 4141 and 4285) and p53 R248W A total of 11 TCRs targeting neoantigens were identified (Table 5). The TCR for patient 4149 could not be determined due to limited T cell availability. R175H / HLA-A * 02:01, p53 R248W / HLA-A * 68:01, p53 R248W / HLA-DPB1 *Identical p53 neoantigen-reactive TCRs from TILs targeting 02:01 (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14 (2019)) were identified in PBL after IVS and 4-1BB / OX40 enrichment in patients 4141, 4266, and 4273. No additional TP53 mutation-reactive T cells were identified in these patients. In contrast, 7 unique p53 TCRs from patient 4285 that were not present in the TIL study were identified. R175H Neoantigen-specific TCRs were identified from PBLs, and TIL-derived TCRs were not found in the PBL population. The functional avidity of the PBL-derived TCR (4285-PBL-TCR) was comparable to that of the TIL-derived TCR (4285-TIL-TCR), and p53 R175H LP was recognized up to 10 ng / mL, and WT p53 R175 No response was observed to LP (Figures 2C-2J). R175H Tracking of neoantigen-specific TCRB clonotypes showed exponential amplification after IVS and 4-1BB / OX40 enrichment compared to starting PBL (Figure 2K). Furthermore, CDR3B sequences from patient 4285 were within the detection limit (2x10 5 Although the frequency was <0.001%) from the reads, it was frequent enough after IVS and 4-1BB / OX40 enrichment to include 4 TCRs in the top 10 across CDR3B clonotypes (Table 5). R248W Neoantigen-specific TCRs were also below the limit of detection in PBLs, but were present in 2.6% (4266-PBL-TCR3) and 7.5% (4266-PBL-TCR2) of 4266-CD8 TP53-TMG-IVS cultures (Figure 2L). R248W Neoantigen-specific TCRs were enriched from 0.002% to 0.017% in 4273-CD4 p53-LP-IVS cultures (Figure 2L). Collectively, the data demonstrate that PBLs can be a source of TCRs reactive to published TP53 mutations that are identical or equivalent to intratumoral TCRs.

[0149] Example 5 This example demonstrates that common HLA-restricted elements and p53 neoepitopes are immunogenic.

[0150] The shortest recognized p53 neoepitopes and corresponding HLA restriction were then assessed. HLA mapping was achieved by transfecting DNA plasmids corresponding to each of the patient's individual HLA alleles into COS7 monkey cell lines (lacking HLA) and pulsing with peptide or co-transfecting with TMG, as previously reported (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019); Deniger et al., Clin. Cancer Res., 24(22):5562-73(2018)). 4141-CD8 TP53-TMG-IVS cultures were HLA-A-restricted as measured by 4-1BB expression. * p53 in the context of 02:01 (highly frequent HLA in the US population) R175H H MTEVVR H C (SEQ ID NO: 96) was specific for the neoepitope (Gonzales et al., Hisp. Health Care Int., 15(4):180-8(2017)) (Figure 3A). HLA-A * 68:01 inhibited p53 recognition by CD8 TP53-TMG-IVS cultures as measured by IFNγ secretion. R248W Neoepitope SSCMGGMN W R (SEQ ID NO: 98) (Figure 3B). This was expected since TCRs from TILs in patients 4141 and 4266 were found in these IVS cultures, where the shortest neo-epitopes and HLA restriction elements had already been established (Malekzadeh et al., J. Clin. Invest. 129(3):1109-14(2019)). Similarly, 4273-PBL-TCR was found in 4273-CD4 p53-LP-IVS cultures, where it bound the p53 TCR known from TIL studies. R248W and HLA-DPB1 *Even though the TCRs differed between PBL and TIL for patient 4285, the 4285-CD4 TP53-TMG-IVS cultures showed the same p53 expression found in the TILs. R175H and HLA-DBR1 * 13:01 combination (Figure 3C). Furthermore, all 4285-PBL-TCRs expressed p53 R175H and HLA-DBR1 * p53 13:01 specific and were all present in 4285-CD4 TP53-TMG-IVS cultures by TCRB sequencing (Table 5; Fig. 4). R175H Peptides were pulsed onto DCs from patient 4285 and cocultured with TCR-transduced T cells to express the core peptide EVVR H CPHHER (SEQ ID NO: 2) is HLA-DRB1 * We determined that this is the consensus sequence recognized by the 4285-PBL-TCR in the 13:01 relationship (Table 6). In summary, we found the same TCR in TILs in PBLs recognizing the same HLA and shortest p53 neoepitope in three cases, and in one case we found an additional TCR with the same p53 neoantigen specificity as the intratumoral T cells.

[0151] [Table 6]

[0152] Example 6 This example demonstrates that tumor cells process and present p53 neoepitopes on HLA that are recognized by PBL-derived T cells after IVS.

[0153] TP53 mutant-reactive T cells were assessed for their ability to recognize naturally processed and presented antigens expressed on the tumor cell surface. * 02:01 and full length p53 R175Hoverexpressing p53) and the TC#4266 human xenograft tumor cell line ( R248W And HLA-A * 68:01:02 colon cancer) was used as a model. After overnight co-culture, CD8 + T cells upregulated 4-1BB in response to Saos2-R175H and TC#4266 cell lines, respectively (cross-matched cell lines were only marginally activated (Figure 3D)). Thus, TP53 mutation-specific T cells from PBLs express 4-1BB in response to naturally processed and presented p53 neoepitopes. It can recognize tumor cells that have

[0154] Example 7 This example shows the amino acid sequences of the TCRs constructed in Example 4 and named in the header of Table 6. The amino acid sequences of the variable regions of the TCR alpha and beta chains of these TCRs are shown in Table 7. The CDRs are underlined.

[0155] [Table 7-1]

[0156] [Table 7-2]

[0157] [Table 7-3]

[0158] [Table 7-4]

[0159] [Table 7-5]

[0160] [Table 7-6]

[0161] [Table 7-7]

[0162] [ka]

[0163] The sequence of TCR 4285-PBL-TCR1 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:6), the second underlined region is CDR2 beta (SEQ ID NO:7), the third underlined region is CDR3 beta (SEQ ID NO:8), the fourth underlined region is CDR1 alpha (SEQ ID NO:3), the fifth underlined region is CDR2 alpha (SEQ ID NO:4), and the sixth underlined region is CDR3 alpha (SEQ ID NO:5). The bolded region is the linker (SEQ ID NO:94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:93), and the second italicized region is the alpha chain constant region (SEQ ID NO:91). The beta chain variable region (SEQ ID NO:10) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:9) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:12) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:11) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0164] A variant of 4285-PBL-TCR1 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 180. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 178. The full-length alpha chain of the variant is shown in SEQ ID NO: 179.

[0165] Another variant of 4285-PBL-TCR1 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 205. The full length beta chain of the variant is shown in SEQ ID NO: 206.

[0166] The predicted variable region mature sequences of 4285-PBL-TCR1 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 142. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 160. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 143. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 161.

[0167] [ka]

[0168] The sequence of TCR 4285-PBL-TCR2 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO: 17), the second underlined region is CDR2 beta (SEQ ID NO: 18), the third underlined region is CDR3 beta (SEQ ID NO: 19), the fourth underlined region is CDR1 alpha (SEQ ID NO: 14), the fifth underlined region is CDR2 alpha (SEQ ID NO: 15), and the sixth underlined region is CDR3 alpha (SEQ ID NO: 16). The bolded region is the linker (SEQ ID NO: 94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO: 93), and the second italicized region is the alpha chain constant region (SEQ ID NO: 91). The beta chain variable region (SEQ ID NO: 21) comprises the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:20) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:23) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:22) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0169] A variant of 4285-PBL-TCR2 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 183. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 181. The full-length alpha chain of the variant is shown in SEQ ID NO: 182.

[0170] Another variant of 4285-PBL-TCR2 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 207. The full length beta chain of the variant is shown in SEQ ID NO: 208.

[0171] The predicted variable region mature sequences of 4285-PBL-TCR2 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 144. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 162. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 145. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 163.

[0172] [ka]

[0173] The sequence of TCR 4285-PBL-TCR3 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:28), the second underlined region is CDR2 beta (SEQ ID NO:29), the third underlined region is CDR3 beta (SEQ ID NO:30), the fourth underlined region is CDR1 alpha (SEQ ID NO:25), the fifth underlined region is CDR2 alpha (SEQ ID NO:26), and the sixth underlined region is CDR3 alpha (SEQ ID NO:27). The bolded region is the linker (SEQ ID NO:94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:93), and the second italicized region is the alpha chain constant region (SEQ ID NO:91). The beta chain variable region (SEQ ID NO:32) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:31) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:34) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:33) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0174] A variant of 4285-PBL-TCR3 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 186. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 184. The full-length alpha chain of the variant is shown in SEQ ID NO: 185.

[0175] Another variant of 4285-PBL-TCR3 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 209. The full length beta chain of the variant is shown in SEQ ID NO: 210.

[0176] The predicted variable region mature sequences of 4285-PBL-TCR3 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 146. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 164. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 147. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 165.

[0177] [ka]

[0178] The sequence of TCR 4285-PBL-TCR5 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:39), the second underlined region is CDR2 beta (SEQ ID NO:40), the third underlined region is CDR3 beta (SEQ ID NO:41), the fourth underlined region is CDR1 alpha (SEQ ID NO:36), the fifth underlined region is CDR2 alpha (SEQ ID NO:37), and the sixth underlined region is CDR3 alpha (SEQ ID NO:38). The bolded region is the linker (SEQ ID NO:94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:93), and the second italicized region is the alpha chain constant region (SEQ ID NO:91). The beta chain variable region (SEQ ID NO:43) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:42) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:45) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:44) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0179] A variant of 4285-PBL-TCR5 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 189. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 187. The full-length alpha chain of the variant is shown in SEQ ID NO: 188.

[0180] Another variant of 4285-PBL-TCR5 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 211. The full length beta chain of the variant is shown in SEQ ID NO: 212.

[0181] The predicted variable region mature sequences of 4285-PBL-TCR5 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 148. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 166. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 149. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 167.

[0182] [ka]

[0183] The sequence of TCR 4285-PBL-TCR6 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:50), the second underlined region is CDR2 beta (SEQ ID NO:51), the third underlined region is CDR3 beta (SEQ ID NO:52), the fourth underlined region is CDR1 alpha (SEQ ID NO:47), the fifth underlined region is CDR2 alpha (SEQ ID NO:48), and the sixth underlined region is CDR3 alpha (SEQ ID NO:49). The bolded region is the linker (SEQ ID NO:94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:93), and the second italicized region is the alpha chain constant region (SEQ ID NO:91). The beta chain variable region (SEQ ID NO:54) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:53) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:56) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:55) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0184] A variant of 4285-PBL-TCR6 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 192. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 190. The full-length alpha chain of the variant is shown in SEQ ID NO: 191.

[0185] Another variant of 4285-PBL-TCR6 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 213. The full length beta chain of the variant is shown in SEQ ID NO: 214.

[0186] The predicted variable region mature sequences of 4285-PBL-TCR6 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 150. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 168. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 151. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 169.

[0187] [ka]

[0188] The sequence of TCR 4285-PBL-TCR7 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:61), the second underlined region is CDR2 beta (SEQ ID NO:62), the third underlined region is CDR3 beta (SEQ ID NO:63), the fourth underlined region is CDR1 alpha (SEQ ID NO:58), the fifth underlined region is CDR2 alpha (SEQ ID NO:59), and the sixth underlined region is CDR3 alpha (SEQ ID NO:60). The bolded region is the linker (SEQ ID NO:94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:93), and the second italicized region is the alpha chain constant region (SEQ ID NO:91). The beta chain variable region (SEQ ID NO:65) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:64) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:67) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:66) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0189] A variant of 4285-PBL-TCR7 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 195. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 193. The full-length alpha chain of the variant is shown in SEQ ID NO: 194.

[0190] Another variant of 4285-PBL-TCR7 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 215. The full length beta chain of the variant is shown in SEQ ID NO: 216.

[0191] The predicted variable region mature sequences of 4285-PBL-TCR7 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 152. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 170. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 153. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 171.

[0192] [ka]

[0193] The sequence of TCR 4285-PBL-TCR9 isolated from patient 4285 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO: 72), the second underlined region is CDR2 beta (SEQ ID NO: 73), the third underlined region is CDR3 beta (SEQ ID NO: 74), the fourth underlined region is CDR1 alpha (SEQ ID NO: 69), the fifth underlined region is CDR2 alpha (SEQ ID NO: 70), and the sixth underlined region is CDR3 alpha (SEQ ID NO: 71). The bolded region is the linker (SEQ ID NO: 94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO: 93), and the second italicized region is the alpha chain constant region (SEQ ID NO: 91). The beta chain variable region (SEQ ID NO: 76) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:75) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:78) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:77) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0194] A variant of 4285-PBL-TCR9 with a wild-type alpha chain signal peptide is shown in SEQ ID NO: 198. The variant contains an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 196. The full-length alpha chain of the variant is shown in SEQ ID NO: 197.

[0195] Another variant of 4285-PBL-TCR9 contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 217. The full length beta chain of the variant is shown in SEQ ID NO: 218.

[0196] The predicted variable region mature sequences of the 4285-PBL-TCR9 alpha and beta chains without the N-terminal signal peptide are shown in Table 7. The predicted alpha chain variable region for the mature sequence without the N-terminal signal peptide is shown in SEQ ID NO: 154. The predicted full length alpha chain (including the alpha chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 172. The predicted beta chain variable region without the N-terminal signal peptide is shown in SEQ ID NO: 155. The predicted full length beta chain (including the beta chain variable region and constant region) without the N-terminal signal peptide is shown in SEQ ID NO: 173.

[0197] Example 8 This example describes the CD8 T cells detected after co-culture with the mutant p53-Y220C peptide from a tumor sample from patient 4259. + 4-1BB + Demonstration of T cell frequency.

[0198] CD8 from 4259-F1 tumor fragment cultures + After sorting of T cells, T cell clones were prepared by limiting dilution. 24 cultures were cultured in DMSO (peptide vehicle), WT p53-Y220 peptide,

[0199] [ka]

[0200] or MUTp53-Y220C peptide

[0201] [ka]

[0202] T2 tumor cells pulsed with HLA-A * After overnight incubation, cells were stained for CD3, CD8, and 4-1BB and then analyzed by flow cytometry. + 4-1BB + The frequency of T cells is shown in Figure 5. As shown in Figure 5, reactive T cell clones were obtained.

[0203] Example 9 This example demonstrates the isolation of the p53-Y220C neoantigen-reactive T cell receptor 4259-F1-TCR.

[0204] p53-Y220C and HLA-A * It was difficult to determine the sequence of the TCR with specificity for 02:01 (from fragment culture F1 of patient 4259 in Example 8). The sequence could not be determined using classical single-cell PCR techniques. Two limitations of single-cell PCR techniques are that (1) it sequences only a short portion of the TCR gene surrounding the hypervariable CDR3 region (allowing researchers to infer the remaining TCR using less polymorphic regions of the TCR, e.g., the variable family), and (2) only one sequence can be present to obtain a functional sequencing readout by Sanger sequencing. These problems were circumvented by generating T cell clones by suppressing dilution and then sequencing the full-length TCR alpha and TCR beta genes as expressed mRNA transcripts by 5'RACE. p53-Y220C and HLA-A were then sequenced. *We determined that T cell clonotypes with specificity for 02:01 express two TCR alpha chains and two TCR beta chains. This is in contrast to most T cells, which express only one TCR alpha and one TCR beta. Only one of the TCR beta chains is functional because the other has a frameshift resulting in a premature stop codon. Furthermore, this TCR beta chain (reported by single-cell PCR and (low-resolution) Adaptive Biotechnologies TCR Survey sequencing of the TRBV7-9 family) is functional in the TRBV7-9 family. * Not the 01 family, but the TRBV7-9 * The 03 family had a non-conservative amino acid substitution (N or D at position 26) between the two variable chains, which indicates that TRBV7-9 * The results show that the selection of 03 was useful (see Figure 9). There were two functional TCR alpha chains identified, only one of which was correctly paired with a functional TCR beta, and was associated with p53-Y220C and HLA-A * Specificity given to 02:01.

[0205] The TCR pair was reconstructed and cloned into a retroviral vector. The second amino acid residue was changed to alanine (A) to have a stronger Kozak sequence for highly efficient translation. The TCR α chain constant region was replaced with a cysteine-substituted, LVL-modified mouse α chain constant region. The TCR β chain constant region was replaced with a cysteine-substituted mouse β chain constant region.

[0206] [ka]

[0207] The sequence of p53-Y220C neoantigen-reactive TCR 4259-F1-TCR isolated from patient 4259 is shown immediately above. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:83), the second underlined region is CDR2 beta (SEQ ID NO:84), the third underlined region is CDR3 beta (SEQ ID NO:85), the fourth underlined region is CDR1 alpha (SEQ ID NO:80), the fifth underlined region is CDR2 alpha (SEQ ID NO:81), and the sixth underlined region is CDR3 alpha (SEQ ID NO:82). The bolded region is the linker (SEQ ID NO:94). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:93), and the second italicized region is the alpha chain constant region (SEQ ID NO:91). The beta chain variable region (SEQ ID NO:87) includes the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:86) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:89) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:88) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0208] A variant of 4259-F1-TCR comprising an alpha chain with a wild-type signal peptide is shown in SEQ ID NO: 201. The variant comprises an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 199. The full-length alpha chain of the variant is shown in SEQ ID NO: 200.

[0209] Another variant of 4259-F1-TCR contains a beta chain variable region (with wild type signal peptide) as set forth in SEQ ID NO: 219. The full length beta chain of the variant is shown in SEQ ID NO: 220.

[0210] The amino acid sequences of the alpha and beta chain variable regions of 4259-F1-TCR, without the N-terminal signal peptide, are shown in Table 8. The CDRs are underlined. The predicted full length alpha chain (including the alpha chain variable and constant regions) without the N-terminal signal peptide is shown in SEQ ID NO: 174. The predicted full length beta chain (including the beta chain variable and constant regions) without the N-terminal signal peptide is shown in SEQ ID NO: 175.

[0211] [Table 8]

[0212] Example 10 This example demonstrates that the 4259-F1-TCR of Example 9 specifically recognizes the mutated p53-Y220C peptide and not the corresponding WT peptide.

[0213] 4259-F1-TCR of Example 9 was transduced into donor peripheral blood T cells and then gradually Concentration of WT p53-Y220 peptide

[0214] [ka]

[0215] or MUT p53-Y220C peptide

[0216] [ka]

[0217] T2 tumor cells (HLA-A * After overnight incubation, the co-culture supernatants were analyzed for interferon gamma secretion by ELISA. The results are shown in Figure 6.

[0218] Example 11 In this example, 4259-F1-TCR was found to be p53-Y220C (HLA-A * We demonstrate that the antibody specifically recognizes tumor cells expressing the IL-1 marker (presented by 02:01).

[0219] T cells expressing either no TCR (non-transduced), p53-R175H-specific TCR, or 4259-F1-TCR of Example 9 were transduced with HLA-A * 02:01, with or without expression of p53-R175H or p53-Y220C, were co-cultured with tumor cells. After overnight incubation, cells were stained for CD3, CD8, and 4-1BB and then analyzed by flow cytometry. + 4-1BB + The frequency of T cells is shown in FIG.

[0220] Example 12 This example demonstrates the isolation and specific reactivity of a TCR from patient 4141.

[0221] A summary of the treatment of patients with p53 mutation-responsive TILs is shown in Table 9.

[0222] [Table 9]

[0223] Autologous APCs were transfected with TMG encoding an irrelevant mutation, the WT p53 sequence, or a mutant p53 sequence containing R175H. Medium alone, and PMA and ionomycin were negative and positive controls, respectively. The following day, TILs from patient 4141 (fragment culture 12) were cocultured with TMG-transfected APCs overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. -1BB expression was determined from lymphocytes → live cells (PI negative) → CD3+ (T cells) → CD4 - CD8 + The results were evaluated by flow cytometry after gating on .

[0224] Cos7 cells (2.5x10 per well 4) were plated into wells of flat-bottom 96-well plates. The next day, cells were co-transfected with individual HLA alleles from patient 4141 and either no additional genes, WT TP53 TMG, or mutant TP53 TMG containing the p53-R175H sequence. TILs with specificity for p53-R175H from patient 4141 (fragment culture 12) were co-cultured the next day with transfected Cos7 cells and incubated overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. The results are shown in Figure 11.

[0225] Mock (no TCR) or 4141-TCR1a2-expressing T cells were injected into T2 tumor cells (HLA-A * T2 cells were co-cultured with T2 cells expressing the WT p53-R175 peptide HMTEVVRRC (SEQ ID NO: 95) or the mutant p53-R175H peptide HMTEVVRHC (SEQ ID NO: 96) for 2 h at 37° C. The cells were pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) peptides consisting of WT p53-R175 peptide HMTEVVRRC (SEQ ID NO: 95) or mutant p53-R175H peptide HMTEVVRHC (SEQ ID NO: 96) for 2 h at 37° C. Media alone, PMA and ionomycin were negative and positive controls, respectively. Co-culture was performed overnight at 37° C. IFN-γ secretion was assessed by ELISA. The results are shown in FIG. 12.

[0226] T cells expressing 4141-TCR1a2 were cocultured with Saos2 cells (p53-NULL and HLA-A) that were either unmanipulated or engineered to overexpress the full-length p53-R175H protein. * 02:01+) at 37°C overnight. Secretion inhibitors (monensin and brefeldin A) were added to the co-culture to trap cytokines in T cells. After 6 hours of co-culture, cells were fixed and permeabilized, then stained for IL-2, CD107a, IFN-γ, and tumor necrosis factor-α (TNFα). Co-cultures were analyzed using flow cytometry based on the lymphocyte gate. The results are shown in Figure 13.

[0227] The sequence of TCR 4141-TCR1a2 isolated from patient 4141 is shown below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO: 131), the second underlined region is CDR2 alpha (SEQ ID NO: 132), the third underlined region is CDR3 alpha (SEQ ID NO: 133), the fourth underlined region is CDR1 beta (SEQ ID NO: 134), the fifth underlined region is CDR2 beta (SEQ ID NO: 135), and the sixth underlined region is CDR3 beta (SEQ ID NO: 136). The bolded region is the linker (SEQ ID NO: 94). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO: 91), and the second italicized region is the beta chain constant region (SEQ ID NO: 93). The alpha chain variable region (SEQ ID NO: 137) includes the sequence starting from the amino terminus and ending just before the start of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 138) contains the sequence starting immediately after the linker and ending just before the start of the beta chain constant region. The full length alpha chain (SEQ ID NO: 139) contains the sequence starting at the amino terminus and ending just before the start of the linker. The full length beta chain (SEQ ID NO: 140) contains the sequence starting immediately after the linker and ending at the carboxy terminus.

[0228] Cancer-reactive T cells were identified as described below. TCRs were isolated as described below. TCR name:4141-TCR1a2 Recognizing p53 mutation: R175H Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: TILs from the 4141 infusion bag, p53mutTMG and Co-culture and selection of CD8+41BB+ T cells Methods for identifying TCR: For the alpha chain, single-cell RT-PCR followed by TA TOPO cloning kit (Thermo Fisher Scientific, Waltham, MA). TCR Orientation: Alpha-Beta Expression vector: SB transposon

[0229] [ka]

[0230] A variant of 4141-TCR1a2 that contains a beta chain with a wild-type signal peptide is shown in SEQ ID NO: 204. The variant contains a beta chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 202. The full length beta chain of the variant is shown in SEQ ID NO: 203.

[0231] Another variant of 4141-TCR1a2 comprising an alpha chain with a wild-type signal peptide is shown in SEQ ID NO: 225. The variant comprises a beta chain variable region as set forth in SEQ ID NO: 221. The full length beta chain of the variant is shown in SEQ ID NO: 222. The variant comprises an alpha chain variable region (with a wild-type signal peptide) as set forth in SEQ ID NO: 223. The full length alpha chain of the variant is shown in SEQ ID NO: 224.

[0232] The amino acid sequences of the 4141-TCR1a2 alpha and beta chain variable regions, without the N-terminal signal peptide, are shown in Table 10. The CDRs are underlined. The predicted full length alpha chain (including the alpha chain variable and constant regions), without the N-terminal signal peptide, is shown in SEQ ID NO: 176. The predicted full length beta chain (including the beta chain variable and constant regions), without the N-terminal signal peptide, is shown in SEQ ID NO: 177.

[0233] [Table 10]

[0234] Statistics for 4141-TCR1a2 from patient 4141 are shown in Table 11 below.

[0235] [Table 11]

[0236] TCR 4141-TCR1a2 was isolated, expressed in T cells and tested against relevant antigens. A summary of the results is shown in Table 12.

[0237] [Table 12]

[0238] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and to the same extent as if each reference was set forth in its entirety herein.

[0239] In describing the present invention (particularly in relation to the claims which follow), the use of the terms "a" and "an" and "the" and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise stated herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise stated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated. Recitation of ranges of values ​​herein is intended only to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary phrases (e.g., "such as") provided herein is intended only to better illustrate the invention and does not impose limitations on the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0240] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of skill in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those of skill in the art, and the inventors intend that the invention 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. Human p53 Ｒ１７５Ｈ or human p53 Ｙ２２０Ｃ 1. An isolated or purified T cell receptor (TCR) having antigen specificity for the amino acid sequence of: (1) All of SEQ ID NOs: 3 to 8; (2) all of SEQ ID NOs: 14 to 19; (3) all of SEQ ID NOs: 25 to 30; (4) all of SEQ ID NOs: 36 to 41; (5) all of SEQ ID NOs: 47 to 52; (6) all of SEQ ID NOs: 58 to 63; (7) all of SEQ ID NOs: 69 to 74; (8) all of SEQ ID NOs: 80 to 85; or (9) All of SEQ ID NOs: 131 to 136 A TCR comprising the amino acid sequence of

2. (1) both SEQ ID NOs:9 and 10; (2) both SEQ ID NOs: 20 and 21; (3) both SEQ ID NOs: 31 and 32; (4) both SEQ ID NOs: 42 and 43; (5) both SEQ ID NOs: 53 and 54; (6) both of SEQ ID NOs: 64 and 65; (7) both of SEQ ID NOs: 75 and 76; (8) both of SEQ ID NOs: 86 and 87; (9) both of SEQ ID NOs: 137 and 138; (10) both of SEQ ID NOs: 142 and 143; (11) Both of SEQ ID NOs: 144 and 145; (12) both of SEQ ID NOs: 146 and 147; (13) both of SEQ ID NOs: 148 and 149; (14) both of SEQ ID NOs: 150 and 151; (15) both of SEQ ID NOs: 152 and 153; (16) both of SEQ ID NOs: 154 and 155; (17) Both of SEQ ID NOs: 156 and 157; (18) both of SEQ ID NOs: 159 and 158; (19) both of SEQ ID NOs: 178 and 10; (20) both of SEQ ID NOs: 181 and 21; (21) Both of SEQ ID NOs: 184 and 32; (22) both of SEQ ID NOs: 187 and 43; (23) both of SEQ ID NOs: 190 and 54; (24) both of SEQ ID NOs: 193 and 65; (25) both of SEQ ID NOs: 196 and 76; (26) both of SEQ ID NOs: 199 and 87; (27) both of SEQ ID NOs: 137 and 202; (28) both SEQ ID NOs: 9 and 205; (29) both of SEQ ID NOs: 20 and 207; (30) both SEQ ID NOs: 31 and 209; (31) Both of SEQ ID NOs: 42 and 211; (32) both SEQ ID NOs: 53 and 213; (33) both of SEQ ID NOs: 64 and 215; (34) both of SEQ ID NOs: 75 and 217; (35) both of SEQ ID NOs: 86 and 219; (36) both of SEQ ID NOs: 137 and 221; (37) both of SEQ ID NOs: 223 and 202; (38) both of SEQ ID NOs: 223 and 221; (39) both of SEQ ID NOs: 20 and 226; or (40) both of SEQ ID NOs: 181 and 226 The TCR of claim 1, comprising the amino acid sequence:

3. (1) both SEQ ID NOs: 11 and 12; (2) both SEQ ID NOs:22 and 23; (3) both SEQ ID NOs: 33 and 34; (4) both SEQ ID NOs: 44 and 45; (5) both of SEQ ID NOs: 55 and 56; (6) both of SEQ ID NOs: 66 and 67; (7) both of SEQ ID NOs: 77 and 78; (8) both of SEQ ID NOs: 88 and 89; (9) both of SEQ ID NOs: 139 and 140; (10) both of SEQ ID NOs: 160 and 161; (11) Both of SEQ ID NOs: 162 and 163; (12) both of SEQ ID NOs: 164 and 165; (13) both of SEQ ID NOs: 166 and 167; (14) Both of SEQ ID NOs: 168 and 169; (15) both of SEQ ID NOs: 170 and 171; (16) both of SEQ ID NOs: 172 and 173; (17) Both of SEQ ID NOs: 174 and 175; (18) both of SEQ ID NOs: 176 and 177; (19) both of SEQ ID NOs: 179 and 12; (20) both of SEQ ID NOs: 182 and 23; (21) Both of SEQ ID NOs: 185 and 34; (22) both of SEQ ID NOs: 188 and 45; (23) both of SEQ ID NOs: 191 and 56; (24) both of SEQ ID NOs: 194 and 67; (25) both of SEQ ID NOs: 197 and 78; (26) both of SEQ ID NOs: 200 and 89; (27) both of SEQ ID NOs: 139 and 203; (28) both SEQ ID NOs: 11 and 206; (29) both of SEQ ID NOs: 22 and 208; (30) both SEQ ID NOs: 33 and 210; (31) Both of SEQ ID NOs: 44 and 212; (32) both of SEQ ID NOs: 55 and 214; (33) both of SEQ ID NOs: 66 and 216; (34) both of SEQ ID NOs: 77 and 218; (35) both of SEQ ID NOs: 88 and 220; (36) both of SEQ ID NOs: 139 and 222; (37) both of SEQ ID NOs: 224 and 203; (38) both of SEQ ID NOs: 224 and 222; (39) both of SEQ ID NOs: 22 and 227; or (40) both of SEQ ID NOs: 182 and 227 The TCR of claim 1 or 2, comprising the amino acid sequence:

4. Human p53 Ｒ１７５Ｈ The amino acid sequence of claim 1 is SEQ ID NO: 2 or SEQ ID NO:

96. A TCR according to any one of claims 1 to 3.

5. Human p53 Ｙ２２０Ｃ The TCR of any one of claims 1 to 3, wherein the amino acid sequence is SEQ ID NO:

113.

6. The TCR of any one of claims 1 to 4, wherein the TCR does not have antigen specificity for the amino acid sequence of wild-type human p53 of SEQ ID NO:

95.

7. The TCR of any one of claims 1 to 3 and 5, wherein the TCR does not have antigen specificity for the amino acid sequence of wild-type human p53 of SEQ ID NO:

112.

8. An isolated or purified polypeptide comprising a functional portion of the TCR of any one of claims 1 to 3, comprising: (1) All of SEQ ID NOs: 3 to 8; (2) all of SEQ ID NOs: 14 to 19; (3) all of SEQ ID NOs: 25 to 30; (4) all of SEQ ID NOs: 36 to 41; (5) all of SEQ ID NOs: 47 to 52; (6) all of SEQ ID NOs: 58 to 63; (7) all of SEQ ID NOs: 69 to 74; (8) all of SEQ ID NOs: 80 to 85; or (9) All of SEQ ID NOs: 131 to 136 A polypeptide comprising the amino acid sequence of

9. (1) both SEQ ID NOs:9 and 10; (2) both SEQ ID NOs: 20 and 21; (3) both SEQ ID NOs: 31 and 32; (4) both SEQ ID NOs: 42 and 43; (5) both SEQ ID NOs: 53 and 54; (6) both of SEQ ID NOs: 64 and 65; (7) both of SEQ ID NOs: 75 and 76; (8) both of SEQ ID NOs: 86 and 87; (9) both of SEQ ID NOs: 137 and 138; (10) both of SEQ ID NOs: 142 and 143; (11) Both of SEQ ID NOs: 144 and 145; (12) both of SEQ ID NOs: 146 and 147; (13) both of SEQ ID NOs: 148 and 149; (14) both of SEQ ID NOs: 150 and 151; (15) both of SEQ ID NOs: 152 and 153; (16) both of SEQ ID NOs: 154 and 155; (17) Both of SEQ ID NOs: 156 and 157; (18) both of SEQ ID NOs: 159 and 158; (19) both of SEQ ID NOs: 178 and 10; (20) both of SEQ ID NOs: 181 and 21; (21) Both of SEQ ID NOs: 184 and 32; (22) both of SEQ ID NOs: 187 and 43; (23) both of SEQ ID NOs: 190 and 54; (24) both of SEQ ID NOs: 193 and 65; (25) both of SEQ ID NOs: 196 and 76; (26) both of SEQ ID NOs: 199 and 87; (27) both of SEQ ID NOs: 137 and 202; (28) both SEQ ID NOs: 9 and 205; (29) both of SEQ ID NOs: 20 and 207; (30) both SEQ ID NOs: 31 and 209; (31) Both of SEQ ID NOs: 42 and 211; (32) both SEQ ID NOs: 53 and 213; (33) both of SEQ ID NOs: 64 and 215; (34) both of SEQ ID NOs: 75 and 217; (35) both of SEQ ID NOs: 86 and 219; (36) both of SEQ ID NOs: 137 and 221; (37) both of SEQ ID NOs: 223 and 202; (38) both of SEQ ID NOs: 223 and 221; (39) both of SEQ ID NOs: 20 and 226; or (40) both of SEQ ID NOs: 181 and 226 The polypeptide of claim 8 comprising the amino acid sequence:

10. (1) both SEQ ID NOs: 11 and 12; (2) both SEQ ID NOs:22 and 23; (3) both SEQ ID NOs: 33 and 34; (4) both SEQ ID NOs: 44 and 45; (5) both of SEQ ID NOs: 55 and 56; (6) both of SEQ ID NOs: 66 and 67; (7) both of SEQ ID NOs: 77 and 78; (8) both of SEQ ID NOs: 88 and 89; (9) both of SEQ ID NOs: 139 and 140; (10) both of SEQ ID NOs: 160 and 161; (11) Both of SEQ ID NOs: 162 and 163; (12) both of SEQ ID NOs: 164 and 165; (13) both of SEQ ID NOs: 166 and 167; (14) Both of SEQ ID NOs: 168 and 169; (15) both of SEQ ID NOs: 170 and 171; (16) both of SEQ ID NOs: 172 and 173; (17) Both of SEQ ID NOs: 174 and 175; (18) both of SEQ ID NOs: 176 and 177; (19) both of SEQ ID NOs: 179 and 12; (20) both of SEQ ID NOs: 182 and 23; (21) Both of SEQ ID NOs: 185 and 34; (22) both of SEQ ID NOs: 188 and 45; (23) both of SEQ ID NOs: 191 and 56; (24) both of SEQ ID NOs: 194 and 67; (25) both of SEQ ID NOs: 197 and 78; (26) both of SEQ ID NOs: 200 and 89; (27) both of SEQ ID NOs: 139 and 203; (28) both SEQ ID NOs: 11 and 206; (29) both of SEQ ID NOs: 22 and 208; (30) both SEQ ID NOs: 33 and 210; (31) Both of SEQ ID NOs: 44 and 212; (32) both of SEQ ID NOs: 55 and 214; (33) both of SEQ ID NOs: 66 and 216; (34) both of SEQ ID NOs: 77 and 218; (35) both of SEQ ID NOs: 88 and 220; (36) both of SEQ ID NOs: 139 and 222; (37) both of SEQ ID NOs: 224 and 203; (38) both of SEQ ID NOs: 224 and 222; (39) both of SEQ ID NOs: 22 and 227; or (40) both of SEQ ID NOs: 182 and 227 10. The polypeptide of claim 8 or 9, comprising the amino acid sequence:

11. (1) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 3 to 5 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 6 to 8; (2) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 14-16 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 17-19; (3) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 25-27 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 28-30; (4) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 36-38 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 39-41; (5) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 47-49 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 50-52; (6) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 58-60 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 61-63; (7) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 69-71 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 72-74; (8) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 80-82 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 83-85; or (9) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 131 to 133 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 134 to 136. An isolated or purified protein comprising:

12. (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:10; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:20 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:21; (3) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:31 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:32; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:53 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:54; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:64 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:65; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 75 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 76; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 86 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 87; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 138; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 142 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 143; (11) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 144 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 145; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 146 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 147; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 148 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 149; (14) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 150 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 151; (15) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 152 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 153; (16) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 154 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 155; (17) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 156 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 157; (18) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 158 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 159; (19) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 178 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 10; (20) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 181 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 21; (21) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 184 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 32; (22) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 187 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43; (23) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 190 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 54; (24) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 193 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 65; (25) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 196 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 76; (26) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 199 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 87; (27) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 202; (28) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:9 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:205; (29) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 20 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 207; (30) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 31 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 209; (31) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 42 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 211; (32) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:53 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:213; (33) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:64 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:215; (34) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:75 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:217; (35) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 86 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 219; (36) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 137 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221; (37) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 223 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 202; (38) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 223 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 221; (39) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 20 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226; or (40) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 181 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226; The protein of claim 11 .

13. (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:11 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:12; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:22 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:23; (3) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:33 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:34; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:44 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:45; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:55 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:56; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:66 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:67; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:77 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:78; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 88 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 89; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 140; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 160 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 161; (11) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 162 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 163; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 164 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 165; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 166 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 167; (14) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 168 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 169; (15) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 170 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 171; (16) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 172 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 173; (17) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 174 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 175; (18) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 176 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 177; (19) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 179 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 12; (20) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 182 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 23; (21) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 185 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 34; (22) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 188 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 45; (23) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 191 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 56; (24) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 194 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 67; (25) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 197 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 78; (26) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 200 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 89; (27) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 203; (28) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 11 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 206; (29) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 208; (30) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 33 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 210; (31) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:44 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:212; (32) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:55 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:214; (33) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:66 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:216; (34) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:77 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:218; (35) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 88 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 220; (36) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 139 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 222; (37) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 224 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 203; (38) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 224 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 222; (39) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 227; or (40) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 182 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:

227. The protein of claim 11 or 12, comprising:

14. 14. An isolated or purified nucleic acid comprising a nucleotide sequence encoding the TCR of any one of claims 1 to 7, the polypeptide of any one of claims 8 to 10, or the protein of any one of claims 11 to 13.

15. 1. An isolated or purified nucleic acid comprising, from 5' to 3', a first nucleic acid sequence and a second nucleotide sequence, wherein the first and second nucleotide sequences are set forth in SEQ ID NOs: 9 and 10; 10 and 9; 20 and 21; 21 and 20; 31 and 32; 32 and 31; 42 and 43; 43 and 42; 53 and 54; 54 and 53; 64 and 65; 65 and 64; 75, and 76;76 and 75;86 and 87;87 and 86;137 and 138;138 and 137;142 and 143;143 and 142;144 and 145;145 and 144;146 and 147;147 and 146;148 and 149;149 and 148;150 and 151;151 and 150;152 and 153;15 3 and 152; 154 and 155; 155 and 154; 156 and 157; 157 and 156; 159 and 158; 158 and 159; 178 and 10; 10 and 178; 181 and 21; 21 and 181; 184 and 32; 32 and 184; 187 and 43; 43 and 187; 190 and 54; 54 and 190; 193 and 65; 65 and 193; 196 and 76; 76 and 196; 199 and 87; 87 and 199; 137 and 202; 202 and 137; 9 and 205; 205 and 9; 20 and 207; 207 and 20; 31 and 209; 209 and 31; 42 and 211; 211 and 42; 53 and 213; 213 and 53; 64 and 215 A nucleic acid encoding the amino acid sequences of: 215 and 64; 75 and 217; 217 and 75; 86 and 219; 219 and 86; 137 and 221; 221 and 137; 223 and 202; 202 and 223; 223 and 221; 221 and 223; 20 and 226; 226 and 20; 181 and 226; or 226 and 181.

16. 16. The isolated or purified nucleic acid of claim 15, further comprising a third nucleotide sequence inserted between the first nucleotide sequence and the second nucleotide sequence, said third nucleotide sequence encoding a cleavable linker peptide.

17. 17. The isolated or purified nucleic acid of claim 16, wherein the cleavable linker peptide comprises the amino acid sequence of SEQ ID NO:

94.

18. 20. The isolated or purified nucleic acid of claim 17, encoding an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 24, 35, 46, 57, 68, 79, 90, 141, 180, 183, 186, 189, 192, 195, 198, 201, 204, 225, 228, and 229.

19. A recombinant expression vector comprising the nucleic acid of any one of claims 14 to 18.

20. 20. The recombinant expression vector of claim 19, which is a transposon vector or a lentiviral vector.

21. 21. An isolated or purified TCR, polypeptide, or protein encoded by the nucleic acid of any one of claims 14 to 18 or the vector of claim 19 or 20.

22. 21. An isolated or purified TCR, polypeptide or protein resulting from expression of the nucleic acid of any one of claims 14 to 18 or the vector of claim 19 or 20 in a cell.

23. A method for producing a host cell expressing a TCR having antigen specificity for a peptide of SEQ ID NO: 2, 96 or 113, comprising contacting the cell with a vector of claim 19 or 20 under conditions that allow introduction of the vector into the cell.

24. An isolated or purified host cell comprising a nucleic acid according to any one of claims 14 to 18, or a recombinant expression vector according to claim 19 or 20.

25. 25. The host cell of claim 24, wherein the cell is a human lymphocyte.

26. 25. The method of claim 24, 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, and a natural killer (NK) cell. host cells.

27. 27. An isolated or purified cell population comprising a host cell according to any one of claims 24 to 26.

28. 27. A method for producing a TCR of any one of claims 1 to 7, 21 or 22, a polypeptide of any one of claims 8 to 10, 21 or 22, or a protein of any one of claims 11 to 13, 21 or 22, comprising culturing a host cell of any one of claims 24 to 26, or a population of host cells of claim 27, such that the TCR, polypeptide or protein is produced.

29. 27. A pharmaceutical composition comprising: (a) a TCR of any one of claims 1 to 7, 21 or 22, a polypeptide of any one of claims 8 to 10, 21 or 22, a protein of any one of claims 11 to 13, 21 or 22, a nucleic acid of any one of claims 14 to 18, a recombinant expression vector of claim 19 or 20, a host cell of any one of claims 24 to 26, or a cell population of claim 27; and (b) a pharma- ceutical composition comprising a TCR of any one of claims 1 to 7, 21 or 22, a polypeptide of any one of claims 8 to 10, 21 or 22, a protein of any one of claims 11 to 13, 21 or 22, a nucleic acid of any one of claims 14 to 18, a recombinant expression vector of claim 19 or 20, a host cell of any one of claims

30. 1. A method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising cancer cells with a TCR of any one of claims 1-7, 21 or 22, a polypeptide of any one of claims 8-10, 21 or 22, a protein of any one of claims 11-13, 21 or 22, a nucleic acid of any one of claims 14-18, a recombinant expression vector of claim 19 or 20, a host cell of any one of claims 24-26, a population of cells of claim 27, or a pharmaceutical composition of claim 29, thereby forming a complex; and (b) detecting the complex; Including, wherein detection of the complex indicates the presence of cancer in the mammal.

31. 30. A TCR of any one of claims 1 to 7, 21 or 22, a polypeptide of any one of claims 8 to 10, 21 or 22, a protein of any one of claims 11 to 13, 21 or 22, a nucleic acid of any one of claims 14 to 18, a recombinant expression vector of claim 19 or 20, a host cell of any one of claims 24 to 26, a cell population of claim 27, or a pharmaceutical composition of claim 29 for use in inducing an immune response against cancer in a mammal.

32. 30. A TCR of any one of claims 1 to 7, 21 or 22, a polypeptide of any one of claims 8 to 10, 21 or 22, a protein of any one of claims 11 to 13, 21 or 22, a nucleic acid of any one of claims 14 to 18, a recombinant expression vector of claim 19 or 20, a host cell of any one of claims 24 to 26, a cell population of claim 27, or a pharmaceutical composition of claim 29 for use in the treatment or prevention of cancer in a mammal.

33. 33. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of claim 31 or 32, wherein the use comprises administering the cell population to a mammal, and the cell population is autologous to the mammal.

34. 33. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of claim 31 or 32, wherein the use comprises administering the cell population to a mammal, and the cell population is allogeneic to the mammal.

35. The method of claim 30 or any one of claims 31 to 34, wherein the cancer is an epithelial cancer. A TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use.

36. 35. The method of claim 30 or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of any one of claims 31 to 34, wherein the cancer is cholangiocarcinoma, melanoma, colon cancer, rectal cancer, ovarian cancer, endometrial cancer, non-small cell lung cancer (NSCLC), glioblastoma, cervical cancer, head and neck cancer, breast cancer, pancreatic cancer, or bladder cancer.

37. 37. The method of any one of claims 30 and 35-36, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, cell population, or pharmaceutical composition for use of any one of claims 31-36, wherein the cancer is known to comprise a R175H mutation or a Y220C mutation in human p53.

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