T cell receptors recognizing mutated p53

Abstract

To provide further treatment methods of cancer.SOLUTION: Also provided are: an isolated or purified T cell receptor (TCR) having antigenic specificity for human p53 having the G245S mutation; and related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Also disclosed are methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Application No. 62 / 565,383, filed Sep. 29, 2017, which is incorporated by reference in its entirety.

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

[0003] Incorporation by Reference of Electronically Submitted Documents The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously herewith and is identified as follows, is incorporated by reference in its entirety herein: 687,555 byte ASCII (text) file entitled "739980_ST25.txt", dated September 7, 2018. [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] An embodiment of the present invention provides an isolated or purified T cell receptor (TCR) having antigenic specificity for mutant human p53.

[0006] Further embodiments of the invention provide related polypeptides and proteins, as well as nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions related to the TCRs of the invention.

[0007] Further embodiments of the present invention provide methods for detecting the presence of cancer in a mammal and methods for treating or preventing cancer in a mammal. [Brief description of the drawings]

[0008] [Figure 1] Graph showing concentrations of IFN-γ (pg / mL) measured after co-culture of autologous PBLs transduced with one of the indicated TCRs from patient 4127 followed by co-culture with autologous dendritic cells (DCs) pulsed with either WT p53-G245 peptide (wt) (open bars) or mutant p53-G245S peptide (mut) (shaded bars) at the indicated decreasing peptide concentrations (μg / mL). [Diagram 2] Graph showing the percentage of 4-1BB positive cells (% of CD4+mTCRβ+) detected after co-culture of autologous DCs pulsed with either WT p53-G245 peptide (wt) (unshaded bars) or mutant p53-G245S peptide (mut) (shaded bars) at the indicated decreasing peptide concentrations (μg / mL) with autologous PBLs transduced with one of the indicated TCRs from patient 4127. [Diagram 3] Graph showing the percentage of 4-1BB positive cells (% CD4+mTCRβ+) detected after co-culture of autologous DCs pulsed with one of the peptides shown in Table A and autologous PBLs transduced with one of the indicated TCRs from patient 4127. [Figure 4] Graph showing concentration of IFN-γ (pg / mL) measured after co-culture of COS7 cells transduced with one of the indicated HLA alleles and pulsed with DMSO or one of the indicated peptides with TILs from patient 4127 or T cells transduced with one of the indicated TCRs. [Diagram 5]Graph showing the percentage of 4-1BB positive cells (% CD4+mTCRβ+) detected after co-culture of COS7 cells transduced with one of the indicated HLA alleles and pulsed with DMSO or one of the indicated peptides with TILs from patient 4127 or T cells transduced with one of the indicated TCRs. [Figure 6] Graph showing the number of spots per 2×10 effector cells measured after co-culture of allogeneic (DRB3*01:01:01 or DRB3*02:02:01) APCs with TILs from patient 4127 that were (1) electroporated with TMG composed of irrelevant (IRR; white bars with left hatched line), WT p53 (p53-WT; grey bars with left hatched line), or mutant p53 (p53-MUT; grey bars) sequences, or (2) pulsed with peptide vehicle (DMSO; white bars with right hatched line) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-G245 sequence (LP-p53-wt-G245; grey bars with right hatched line) or mutant p53-G245S (LP-p53-mut-G245S; black bars) sequences. [Figure 7] (1) TMGs composed of irrelevant (IRR; white bars with left diagonal lines), WT p53 (p53-WT; grey bars with left diagonal lines), or mutant p53 (p53-MUT; grey bars) sequences were electroporated, or (2) TMGs composed of peptide vehicle (DMSO; white bars with right diagonal lines), or WT p53-G245 sequence (LP-p53-wt-G245; grey bars with right diagonal lines) or mutant p53 (p53-MUT; grey bars) sequences were electroporated. Figure 13 shows the number of spots per 2x10 effector cells measured after co-culture of 4127-TCR1-expressing T cells with allogeneic (DRB3*01:01:01 or DRB3*02:02:01) APCs pulsed with a purified (>95% by HPLC) 25 amino acid peptide composed of the mutated p53-G245S (LP-p53-mut-G245S; black bars) sequence. [Figure 8]Figure 8A is a graph showing IFN-γ secretion (spots / 2x104 cells) measured after co-culture of Cos7 cells transfected with plasmids for TMG1 and the indicated HLA alleles with TILs from the infusion bag used to treat patient 4196. Figure 8B is a graph showing IFN-γ secretion (spots / 2x104 cells) measured after co-culture of autologous DCs pulsed with candidate minimal epitopes (HMTEVVRHC (SEQ ID NO: 530) or (SQHMTEVVRH (SEQ ID NO: 531)) with TILs from the infusion bag used to treat patient 4196. T cells cultured alone served as control. [Figure 9] Figures 9A-9D are graphs showing the percentage of mTCRβ+CD8+41BB+ cells detected after co-culture of cells transduced with one of the following TCRs: AV12 / BV6-1 (A), AV2 / BV6-1 (B), AV6 / BV11 (C), or AV38 / BV10-3 (D) with DCs pulsed with the indicated concentrations (μg / mL) of minimal WT (circles) or mutant (mut) (squares) epitopes. Co-culture of pulsed DCs with untransduced (UT) cells (triangles) served as control. Results shown are representative of two experiments. [Figure 10] 10A-10B are graphs showing IFN-γ secretion (pg / mL) (A) and percentage of 4-1BB positive cells (%, mTCRβ+) (B) measured after co-culture of T2 cells pulsed with the indicated concentrations of minimal mutant epitopes with cells transduced with one of the following TCRs: AV12 / BV6-1 (black triangles), AV6 / BV11 (circles), or AV38 / BV10-3 (squares). Co-culture of pulsed T2 cells with untransduced (UT) cells (white triangles) served as a control. [Figure 11]Figure 1 shows the number of IFN-γ positive spots per 2x104 effector cells detected after co-culture of TCR-transduced cells with the indicated target cell lines. Effector cells were transduced with AV12 / BV6-1 (black bars), AV6 / BV11 (striped bars), or AV38 / BV10-3 (gray bars). HLA-A*02:01 transduced target cells are indicated by LS123-A2, CCRF-CEM-A2, and AU-565-A2. Peptide pulsed target cells are indicated by (+). Non-peptide pulsed target cells are indicated by (-). Non-transduced effector cells (unshaded bars) co-cultured with target cell lines were used as controls. [Figure 12] Graph showing the number of IFN-γ positive spots per 2 × 104 effector cells measured after co-culture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars) or mutant p53 (p53-MUT; black bars) sequences with TIL fragments F1 to F24 (n = 24) from patient 4238. [Figure 13] Graph showing the number of IFN-γ positive spots per 2×104 effector cells measured after co-culture of autologous APCs pulsed with peptide vehicle (DMSO; white bars) or a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence with TIL fragments F1-F24 (n=24) from patient 4238. [Figure 14] Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars) or mutant p53 (p53-MUT; black bars) sequences with TIL fragments F1 to F24 (n=24) from patient 4238. [Figure 15]Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments F1-F24 (n=24) from patient 4238 with autologous APCs pulsed with peptide vehicle (DMSO; white bars) or a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Figure 16] Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of TIL fragments (F1-F24; n=24) derived from patient 4253 with autologous APCs that were (1) pulsed with peptide vehicle (DMSO; white bars), (2) pulsed with a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence, (3) electroporated with irrelevant TMG (TMG-IRR; grey hatched bars), or (4) electroporated with p53-mut-TMG containing the mutant p53-R248W sequence. [Figure 17] Graph showing the number of IFN-γ positive spots per 2 × 104 effector cells measured after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4273 with autologous APCs electroporated with TMGs composed of irrelevant (IRR; white bars), WT p53 (p53-WT; grey bars), or mutant p53 (p53-MUT; black bars) sequences (TMG). [Figure 18] Graph showing the percentage of CD4+4-1BB+ cells detected after coculture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars), WT p53 (p53-WT; gray bars), or mutant p53 (p53-MUT; black bars) sequences with TIL fragments F1 to F24 (n=24) from patient 4273. [Figure 19]Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4273 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence. [Figure 20] Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4273 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence. [Figure 21] Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of autologous PBLs from patient 4149 (transplanted 4149-TCRa2b1 or 4149-TCRa2b2) with autologous APCs (1) electroporated with TMG composed of irrelevant (IRR; black bars with right hatched line), WT p53 (p53-wt-TMG; black bars with left hatched line), or mutant p53 (p53-mut-TMG; gray bars) sequences, or (2) pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-Y220 sequence (LP-p53-Y220-WT; black bars with horizontal line) or mutant p53-Y220C (LP-p53-Y220C-MUT; black bars) sequences. Phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono) were positive controls (grey bars). [Figure 22]Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of autologous PBL from patient 4149 transferred with TCR (4149-TCRa2b1 or 4149-TCRa2b2) with autologous APCs (1) electroporated with TMG composed of irrelevant (IRR; black bars with right hatched line), WT p53 (p53-wt-TMG; black bars with left hatched line), or mutant p53 (p53-mut-TMG; gray bars) sequences, or (2) pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptide composed of WT p53-Y220 sequence (LP-p53-Y220-WT; black bars with horizontal line) or mutant p53-Y220C (LP-p53-Y220C-MUT; black bars). PMA and Iono were positive controls (grey bars). [Figure 23] Graph showing the percentage of 4-1BB+ cells (% of CD4+ T cells) detected after co-culture of autologous DCs pulsed with one of the peptides in Table F with TILs from patient 4149. [Figure 24] Graph showing IFN-γ secretion (pg / mL) after co-culture of Cos7 cells co-transfected with individual HLA alleles + / - TMG with 4149-TCRa2b2 transferred T cells. Cells not transfected with TMG were pulsed with p53Y220C 15mer peptide. Pulsed target cells are shown by shaded bars. TMG transfected target cells are shown by unshaded bars. [Diagram 25] Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments (F2 and F24) derived from patient 4213 with autologous APCs pulsed with peptide vehicle (DMSO; white bars) or a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Figure 26]Graph showing the number of IFN-γ positive spots per 2×104 effector cells measured after co-culture of CD4+ T cells from patient 4213 with autologous APCs pulsed with peptide vehicle (DMSO; white bars) or a purified (>95% by HPLC) 25 amino acid peptide composed of mutant p53-R248Q (LP-p53-R248Q-MUT; black bars). IFN-γ secretion is shown by white bars. 4-1BB expression is shown by black bars. [Figure 27] Graph showing the number of IFN-γ positive spots per 2 × 104 effector cells measured after co-culture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars), WT p53 (p53-WT; grey bars), or mutant p53 (p53-MUT; black bars) sequences with TIL fragments (F1-F24, n=24) derived from patient 4268. [Figure 28] Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4268 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Figure 29] Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4268 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Diagram 30]Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4268 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Diagram 31] Graph showing the number of IFN-γ positive spots per 2 × 104 effector cells measured after co-culture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars), WT p53 (p53-WT; grey bars), or mutant p53 (p53-MUT; black bars) sequences with TIL fragments (F1-F24, n=24) derived from patient 4266. [Diagram 32] Graph showing the percentage of CD8+4-1BB+ cells detected after coculture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars), WT p53 (p53-WT; gray bars), or mutant p53 (p53-MUT; black bars) sequences with TIL fragments (F1-F24, n=24) derived from patient 4266. [Diagram 33] Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4266 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence. [Diagram 34]Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments (F1-F24, n=24) derived from patient 4266 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R248 sequence (LP-p53-R248-WT; grey bars) or mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence. [Diagram 35] 1 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: 535); SP|P04637-3|P53_HUMAN (SEQ ID NO: 536); SP|P04637-4|P53_HUMAN (SEQ ID NO: 537); SP|P04637-5|P53_HUMAN (SEQ ID NO: 538); SP|P04637-6|P53_HUMAN (SEQ ID NO: 539); SP|P04637-7|P53_HUMAN (SEQ ID NO: 540); SP|P04637-8|P53_HUMAN (SEQ ID NO: 541); and SP|P04637-9|P53_HUMAN (SEQ ID NO: 542). [Diagram 36] Graph showing the percentage of 4-1BB positive cells (% mTCRβ+) detected after co-culture of 4127-O37-TCR transduced T cells with autologous APCs pulsed with decreasing concentrations of a 25 amino acid peptide corresponding to the sequence of WT (white circles) or mutant (black squares) p53-G245S. [Figure 37] Figures 37A-37C are graphs showing the percentage of 4-1BB positive cells (% of mTCRβ+) (left y-axis; hatched bars) and IFN-γ (pg / mL) (right y-axis; black bars) measured after co-culture of COS7 cells co-transfected with one of the indicated HLA alleles and pulsed with the 25 amino acid p53-G245S peptide with T cells transduced with 4127-O37-TCR (A), 4127-TP53-G245S-TCR1 (B), or 4127-O102-TCR (C). [Figure 38]Graphs 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 APCs transfected with TMG encoding an irrelevant mutation (TMG-IRR), a WT p53 sequence (TP53-wt-TMG), or a mutant p53 sequence containing R175H (TP53-mut-TMG). Medium alone, and PMA and ionomycin were negative and positive controls, respectively. [Figure 39] Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of Cos7 cells co-transfected with the indicated HLA allele and no additional genes (HLA only; white bars), WT TP53 TMG (grey hatched bars), or mutant TP53 TMG containing the p53-R175H sequence (black bars) with TILs from patient 4141 (fragment culture 12). [Diagram 40] Graph showing IFN-γ concentrations (pg / mL) measured after co-culture of mock (no TCR) or 4141-TCR1a2 expressing T cells with T2 tumor cells (expressing HLA-A*02:01). 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). [Diagram 41]Graph showing the percentage of cells positive for expression of one of the indicated markers after co-culture of Saos2 cells (p53-NULL and HLA-A*02:01+) that were either not engineered (unshaded bars) or made to overexpress full-length p53-R175H protein (shaded bars) with T cells expressing 4141-TCR1a2. Data are mean ± SEM (n=3). Two-tailed Student's t-tests were performed for each cytokine between the two cell lines for statistical analysis (***p<0.001). [Diagram 42] Graph showing the percentage of CD4+4-1BB positive cells detected after co-culture of TILs (fragment culture 6 from patient 4259) with autologous APCs (1) electroporated with TMG composed of irrelevant (TMG-IRR), WT p53 (TMG-p53-WT), or mutant p53 (TMG-p53-MUT) sequences, or (2) pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-Y220 sequence (LP-p53-Y220-WT) or mutant p53-Y220C (LP-p53-Y220C-MUT) sequence. [Diagram 43] Graph showing the percentage of CD4+4-1BB positive cells detected after co-culture of TIL fragment cultures derived from patient 4259 (#6) with autologous APCs pulsed with decreasing concentrations of a 25 amino acid peptide corresponding to the WT p53 sequence (white circles) or mutant p53-Y220C (black squares) for 2 hours at 37°C. [Diagram 44] Graph showing the percentage of CD4+4-1BB positive cells detected after co-culture of TILs from patient 4259 with autologous APCs pulsed with DMSO, WT p53-Y220 peptide, or mutant p53-Y220C peptide. [Diagram 45]Graph showing the percentage of 4-1BB positive cells (% of CD4+) detected after co-culture of TIL fragment culture #6 from patient 4259 with Cos7 cells co-transfected with the indicated HLA alleles from patient 4259 and pulsed with DMSO (white bars) or p53-Y220C peptide (black bars). [Diagram 46] Graph showing percentage of 4-1BB+ cells detected after co-culture of effector T cells (105) expressing mock (no TCR; white bars) or p53-Y220C-specific TCR (4259-F6-TCR; black bars) with TC#4259 target cells (endogenously expressing p53-Y220C and HLA-DRB1*04:01:01). TC#4259 cells were incubated with nothing, W6 / 32 pan-HLA class I-specific blocking antibody, IVA12 pan-HLA class II-specific blocking antibody, or mutant p53-Y220C peptide. Media only (no TC) and PMA and ionomycin were negative and positive controls, respectively. Data are mean ± SEM (n=3). For statistical analysis, two-tailed Student's t-test was performed between groups as indicated (**p<0.01 and ***p<0.001). [Figure 47] Graph showing the number of IFN-γ positive spots per 2 × 104 effector cells measured after co-culture of autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars), WT p53 (p53-WT; grey bars), or mutant p53 (p53-MUT; black bars) sequences with TIL fragments (F1-F22 and F24, n=23) derived from patient 4285. [Figure 48]Graph showing the number of IFN-γ positive spots per 2×10 effector cells measured after co-culture of TIL fragments (F1-F22 and F24, n=23) from patient 4285 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R175 sequence (LP-p53-R175-WT; grey bars) or mutant p53-R175H (LP-p53-R175H-MUT; black bars) sequence. [Figure 49] Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of TIL fragments (F1-F22 and F24, n=23) derived from patient 4285 with autologous APCs electroporated with TMG composed of irrelevant (IRR; white bars), WT p53 (p53-WT; gray bars), or mutant p53 (p53-MUT; black bars) sequences. [Figure 50] Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of TIL fragments (F1-F22 and F24, n=23) derived from patient 4285 with autologous APC pulsed with peptide vehicle (DMSO; white bars) or purified (>95% by HPLC) 25 amino acid peptides composed of the WT p53-R175 sequence (LP-p53-R175-WT; grey bars) or mutant p53-R175H (LP-p53-R175H-MUT; black bars) sequence. [Figure 51] Graph showing the percentage of 4-1BB+ cells (% of CD4+) detected after co-culture of TIL fragment cultures 4285-F6, 4285-F9, and 4285-F10 with Cos7 cells transfected with the indicated HLA alleles and pulsed with DMSO (peptide vehicle; gray and black hatched bars) or mutant p53-R175H peptide (gray, checkered, and black bars). [Figure 52]Graph showing the percentage of 4-1BB+ cells (% of CD4+) detected after co-culture of 4285-TCR1-transferred T cells with autologous APC pulsed with decreasing concentrations of 25 or 15 amino acid peptides corresponding to the sequence of WT (white circles and squares) or mutant (black circles and squares) p53-R175H. [Figure 53] Graph showing the percentage of 4-1BB+ cells (% of CD8+) detected after co-culture of TILs from patient 4266 with Cos7 cells co-transfected with individual HLA alleles from patient 4266 and pulsed with no peptide (white bars), DMSO (peptide vehicle; grey bars), wild-type p53-R248 peptide (grey hatched bars), or mutant p53-R248W peptide (black bars). Data are mean ± SEM (n=3). [Figure 54] Graph showing the percentage of 4-1BB+ cells (% of CD8+) detected after co-culture of autologous APCs pulsed with peptide vehicle (DMSO; grey bars) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 (hatched grey bars) or mutant p53-R248W (black bars) sequences with T cells expressing mock (no TCR), 4266-TCR1, 4266-TCR2, 4266-TCR3, or 4266-TCR4. Medium alone (white bars) and PMA and ionomycin (checkered bars) were negative and positive controls, respectively. [Figure 55]Graph showing percentage of 4-1BB+ cells (% of CD8+) detected after co-culture of a tumor cell (TC) line (TC#4266) established from a xenograft tumor fragment excised from patient 4266 and then serially passaged in immunocompromised mice with mock (no TCR) or T cells expressing p53-R248W-specific TCRs (4266-TCR2, 4266-TCR3, or 4266-TCR4). TC#4266 cells were incubated with either nothing (black bars), W6 / 32 pan-HLA class I-specific blocking antibody (right grey hatched bars), IVA12 pan-HLA class II-specific blocking antibody (grey bars), or mutant p53-R248W peptide (left grey hatched bars). Medium alone (no TC; white bars) and PMA and ionomycin (grey checkered bars) were negative and positive controls, respectively. [Figure 56] Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of autologous APCs transfected with TMG encoding an irrelevant mutation (gray bars), a wild-type p53 sequence (gray hatched bars), or a mutant p53 sequence containing p53-R248W (black bars) with TILs from patient 4273. Medium alone (white bars) and PMA and ionomycin (checkered bars) were negative and positive controls, respectively. [Figure 57] Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of autologous APCs pulsed with a 25 amino acid peptide corresponding to wild-type (white circles) or mutant (black squares) derived from the p53-R248W neoepitope with TILs from patient 4273. DMSO was the peptide vehicle. [Figure 58]Graph showing percentage of CD4+4-1BB+ cells detected after co-culture of autologous APCs pulsed with a 15 amino acid peptide (bold outline indicates amino acid substitution) derived from the p53-R248W neoepitope overlapping 14 amino acids with TILs from patient 4273. DMSO was the peptide vehicle, medium alone (T cells only) and PMA and ionomycin were controls. The 25 amino acid peptides (wt p53-R248 and mutant p53-R248W) were further controls for the 15 amino acid peptide. The peptides are YMCNSSCMGGMNWRP (SEQ ID NO: 592); MCNSSCMGGMNWRPIL (SEQ ID NO: 594); NSSCMGGMNWRPILT (SEQ ID NO: 595); SSCMGGMNWRPILTI (SEQ ID NO: 596); SCMGGMNWRPILTIITII (SEQ ID NO: 597); CMGGMNWRPILTIIT (SEQ ID NO: 598); MGGMNWRPILTIITL (SEQ ID NO: 599); GGMNWRPILTIITLE (SEQ ID NO: 600); GMNWRPILTIITLED (SEQ ID NO: 601); and MNWRPILTIITLEDS (SEQ ID NO: 602). [Figure 59] Graph showing concentration (pg / mL) of secreted IFN-γ after co-culture of Cos7 cells co-transfected with individual HLA alleles from patient 4273 and either WT (white bars) or mutant (black bars) TP53 TMG, with TILs from patient 4273, with or without p53-R248W neoantigen, respectively. [Figure 60] Graph showing IFN-γ spot counts per 2×104 cells measured after co-culture of autologous APCs pulsed with peptide vehicle (DMSO; grey bars) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 (hatched grey bars) or mutant p53-R248W (black bars) sequences with mock (no TCR) or 4273-TCR1a2 expressing T cells. Media alone (white bars) and PMA and ionomycin (checkered bars) were negative and positive controls, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] 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 mutant or damaged DNA, p53 helps to prevent the development of tumors. Wild-type (WT) (normal) full-length p53 comprises the amino acid sequence of SEQ ID NO:1.

[0010] 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.

[0011] An embodiment of the present invention provides an isolated or purified T cell receptor (TCR) having antigen specificity for mutant 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 therefore 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. For example, when a position is 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 "G245S" indicates that the glycine present at position 245 of SEQ ID NO: 1 has been replaced with a serine. P53 has nine known splice variants. 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. 35. Thus, T cells isolated by the methods 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. When a position is defined by SEQ ID NO:1, the actual position of the amino acid sequence of a particular splice variant of p53 is defined relative to the corresponding position in SEQ ID NO:1, and the position defined by SEQ ID NO:1 may differ from the actual position 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 designated position of the 393 amino acid sequence in SEQ ID NO:1, with the understanding that the actual position in a splice variant may differ.

[0012] In an embodiment of the invention, the TCR has antigen specificity for human p53 having a mutation at position 175, 220, 245, 248, 249, 273, or 282 of SEQ ID NO:1. The TCR may have antigen specificity for human p53 having a mutation at position 175, 220, 245, or 248 of SEQ ID NO: 1. The p53 mutation may be any missense mutation. Thus, the mutation at position 175, 220, 245, 248, 249, 273, or 282 of SEQ ID NO: 1 may be a substitution of the native (WT) amino acid residue present at position 175, 220, 245, 248, 249, 273, or 282 of SEQ ID NO: 1 with any amino acid residue other than the native (WT) amino acid residue present at the particular position under consideration. In an embodiment of the present invention, the TCR has antigen specificity for human p53 having one of the following human p53 mutations: R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273H, R273C, R273L, or R282W. The TCR may have antigen specificity for human p53 having one of the following human p53 mutations: R175H, Y220C, G245S, R248Q, or R248W. For example, the TCR of the present invention may have antigen specificity for a mutant p53 amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 13.

[0013] 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.

[0014] The TCRs of the present invention, including when expressed in cells used for adoptive cell transfer, may provide many 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 present 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 present invention may advantageously successfully treat or prevent mutant p53 positive cancers that do not respond to other types of treatment, e.g., chemotherapy, surgery, or radiation. Furthermore, the TCRs of the present invention may 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 mutant p53 and / or applicable HLA molecules, pulsed with p53 peptides carrying p53 mutations, or combinations thereof). Approximately half of all tumors have mutations in p53, about half of which are missense mutations, with about 30% of the missense mutations occurring at the following "hot spot" residues: R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273C, R273L, R273H, and R282W. Furthermore, the same "hot spot" mutations (e.g., R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273H, R273C, R273L, or R282W) frequently occur in unrelated human tumors (cumulatively, about 30% of p53 missense mutations). Thus, the TCRs of the present invention may increase the number of patients potentially eligible for immunotherapeutic treatment.

[0015] The phrase "antigen specificity" as used herein means that the TCR can specifically bind to and immunologically recognize mutant p53 with high avidity. For example, (a) antigen-negative applicable HLA molecule-positive target cells pulsed with 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 a range defined by any two of the above values) or (b) mutant p53 peptide such that the target cells express mutant p53. When co-cultured with antigen-negative, applicable HLA molecule-positive target cells transfected with a nucleotide sequence encoding 3, approximately 1 x 10 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.

[0016] 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. The negative control may be, for example, (i) T cells expressing the TCR co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with the same concentration of an irrelevant peptide (e.g., some other peptide having a sequence different from the mutant p53 peptide) or (b) antigen-negative applicable HLA molecule-positive target cells into which a nucleotide sequence encoding the irrelevant peptide has been introduced so that the target cells express the irrelevant peptide, or (ii) non-transduced T cells (e.g., from PBMCs not expressing the TCR) co-cultured with (a) antigen-negative applicable HLA molecule-positive target cells pulsed with the same 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. IFN-γ secretion can be measured by methods known in the art, for example, enzyme-linked immunosorbent assay (ELISA).

[0017] 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 such 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] The 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 polypeptide of the TCR of the invention may comprise any amino acid sequence, so long as the TCR has antigen specificity for mutant p53.

[0022] 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 is selected from the group consisting of: (1) all of SEQ ID NOs: 27 to 32; (2) all of SEQ ID NOs: 37 to 42; (3) all of SEQ ID NOs: 47 to 52; (4) all of SEQ ID NOs: 57 to 62; (5) all of SEQ ID NOs: 67 to 72; (6) all of SEQ ID NOs: 77 to 82; (7) all of SEQ ID NOs: 87 to 92; (8) all of SEQ ID NOs: 97 to 102; (9) all of SEQ ID NOs: 107 to 112; (10) all of SEQ ID NOs: 117 to 122; (11) all of SEQ ID NOs: 127 to 132; (12) all of SEQ ID NOs: 137 to 142; (13) (14) all of SEQ ID NOs: 147 to 152; (14) all of SEQ ID NOs: 157 to 162; (15) all of SEQ ID NOs: 167 to 172; (16) all of SEQ ID NOs: 177 to 182; (17) all of SEQ ID NOs: 187 to 192; (18) all of SEQ ID NOs: 197 to 202; (19) all of SEQ ID NOs: 207 to 212; (20) all of SEQ ID NOs: 217 to 222; (21) all of SEQ ID NOs: 227 to 232; (22) all of SEQ ID NOs: 237 to 242; (23) all of SEQ ID NOs: 247 to 252; (24) all of SEQ ID NOs: 257 to 262; (25) All of SEQ ID NOs: 267 to 272; (26) All of SEQ ID NOs: 277 to 282; (27) All of SEQ ID NOs: 287 to 292; (28) All of SEQ ID NOs: 297 to 302; (29) All of SEQ ID NOs: 307 to 312; (30) All of SEQ ID NOs: 317 to 322; (31) All of SEQ ID NOs: 327 to 332; (32) All of SEQ ID NOs: 337 to 342; (33) All of SEQ ID NOs: 347 to 352; (34) All of SEQ ID NOs: 357 to 362; (35) All of SEQ ID NOs: 367 to 372; (36) SEQ ID NOs: 377 to 382 (37) all of SEQ ID NOs: 387-392; (38) all of SEQ ID NOs: 397-402; (39) all of SEQ ID NOs: 407-412; (40) all of SEQ ID NOs: 417-422; (41) all of SEQ ID NOs: 427-432; (42) all of SEQ ID NOs: 437-442; (43) all of SEQ ID NOs: 447-452; (44) all of SEQ ID NOs: 457-462; (45) all of SEQ ID NOs: 467-472; (46) all of SEQ ID NOs: 477-482; or (47) all of SEQ ID NOs: 487-492. Each one of the collection of 47 amino acid sequences described above in this paragraph represents six CDR regions of each of 47 different TCRs having antigen specificity for mutant human p53.The six amino acid sequences in each collection are CDR1α, CDR2α, and CDR3, respectively. α, CDR1β, CDR2β, and CDR3β.

[0023] In an embodiment of the invention, the TCR comprises an α chain variable region amino acid sequence and a β chain variable region amino acid sequence that together comprise one of the collections of CDRs set forth above. In this regard, the TCR may comprise: (1) both of SEQ ID NOs: 33-34; (2) both of SEQ ID NOs: 43-44; (3) both of SEQ ID NOs: 53-54; (4) both of SEQ ID NOs: 63-64; (5) both of SEQ ID NOs: 73-74; (6) both of SEQ ID NOs: 83-84; (7) both of SEQ ID NOs: 93-94; (8) both of SEQ ID NOs: 103-104; (9) both of SEQ ID NOs: 113-114; (10) both of SEQ ID NOs: 123-124; (11) both of SEQ ID NOs: 133-134; (12) both of SEQ ID NOs: 143-144; (13) both of SEQ ID NOs: (14) both SEQ ID NOs: 163-164; (15) both SEQ ID NOs: 173-174; (16) both SEQ ID NOs: 183-184; (17) both SEQ ID NOs: 193-194; (18) both SEQ ID NOs: 203-204; (19) both SEQ ID NOs: 213-214; (20) both SEQ ID NOs: 223-224; (21) both SEQ ID NOs: 233-234; (22) both SEQ ID NOs: 243-244; (23) both SEQ ID NOs: 253-254; (24) both SEQ ID NOs: 263-264; (2 5) both of SEQ ID NOs: 273 to 274; (26) both of SEQ ID NOs: 283 to 284; (27) both of SEQ ID NOs: 293 to 294; (28) both of SEQ ID NOs: 303 to 304; (29) both of SEQ ID NOs: 313 to 314; (30) both of SEQ ID NOs: 323 to 324; (31) both of SEQ ID NOs: 333 to 334; (32) both of SEQ ID NOs: 343 to 344; (33) both of SEQ ID NOs: 353 to 354; (34) both of SEQ ID NOs: 363 to 364; (35) both of SEQ ID NOs: 373 to 374; (36) both of SEQ ID NOs: 383 to 384 (37) both of SEQ ID NOs: 393 to 394; (38) both of SEQ ID NOs: 403 to 404; (39) both of SEQ ID NOs: 413 to 414; (40) both of SEQ ID NOs: 423 to 424; (41) both of SEQ ID NOs: 433 to 434; (42) both of SEQ ID NOs: 443 to 444; (43) both of SEQ ID NOs: 453 to 454; (44) both of SEQ ID NOs: 463 to 464; (45) both of SEQ ID NOs: 473 to 474; (46) both of SEQ ID NOs: 483 to 484; or (47) both of SEQ ID NOs: 493 to 494.Each one of the collections of 47 amino acid sequences mentioned 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.

[0024] The TCRs of the invention may further comprise a constant region. The constant region may be from any suitable species, such as, for example, human or mouse. In an embodiment of the invention, the TCR further comprises a mouse constant region. As used herein, the term "mouse" or "human" refers to a TCR (or a component thereof) of mouse or human origin, respectively, 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), i.e., a TCR (or a component thereof) originating from or once expressed by a mouse T cell or a human T cell, respectively. In an embodiment of the invention, the TCR may comprise a mouse alpha chain constant region and a mouse beta chain constant region. The mouse alpha chain constant region may be modified or unmodified. The modified mouse alpha chain constant region may be, for example, cysteine ​​substituted, LVL modified, or both cysteine ​​substituted and LVL modified, for example, as described in U.S. Patent Application Publication No. 2017 / 0145070. 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 US Patent Application Publication No. 2017 / 0145070. 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: 23 or 24. 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: 25.

[0025] 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.In an embodiment of the invention, the TCR is: (1) both of SEQ ID NOs: 35-36; (2) both of SEQ ID NOs: 45-46; (3) both of SEQ ID NOs: 55-56; (4) both of SEQ ID NOs: 65-66; (5) both of SEQ ID NOs: 75-76; (6) both of SEQ ID NOs: 85-86; (7) both of SEQ ID NOs: 95-96; (8) both of SEQ ID NOs: 105-106; (9) both of SEQ ID NOs: 115-116; (10) both of SEQ ID NOs: 125-126; (11) both of SEQ ID NOs: 135-136; (12) both of SEQ ID NOs: 145-146; (13) both of SEQ ID NOs: 150-152; (14) both of SEQ ID NOs: 165-166; (15) both of SEQ ID NOs: 175-176; (16) both of SEQ ID NOs: 185-186; (17) both of SEQ ID NOs: 195-196; (18) both of SEQ ID NOs: 205-206; (19) both of SEQ ID NOs: 215-216; (20) both of SEQ ID NOs: 225-226; (21) both of SEQ ID NOs: 235-236; (22) both of SEQ ID NOs: 245-246; (23) both of SEQ ID NOs: 255-256; (24) both of SEQ ID NOs: 265-266; (25) both of SEQ ID NOs: 275 to 276; (26) both of SEQ ID NOs: 285 to 286; (27) both of SEQ ID NOs: 295 to 296; (28) both of SEQ ID NOs: 305 to 306; (29) both of SEQ ID NOs: 315 to 316; (30) both of SEQ ID NOs: 325 to 326; (31) both of SEQ ID NOs: 335 to 336; (32) both of SEQ ID NOs: 345 to 346; (33) both of SEQ ID NOs: 355 to 356; (34) both of SEQ ID NOs: 365 to 366; (35) both of SEQ ID NOs: 375 to 376; (36) both of SEQ ID NOs: 385 to 386 (37) both of SEQ ID NOs: 395-396; (38) both of SEQ ID NOs: 405-406; (39) both of SEQ ID NOs: 415-416; (40) both of SEQ ID NOs: 425-426; (41) both of SEQ ID NOs: 435-436; (42) both of SEQ ID NOs: 445-446; (43) both of SEQ ID NOs: 455-456; (44) both of SEQ ID NOs: 465-466; (45) both of SEQ ID NOs: 475-476; (46) both of SEQ ID NOs: 485-486; or (47) both of SEQ ID NOs: 495-496. Each one of the collection of 47 amino acid sequences described above in this paragraph represents the α and β chains of each of 47 different TCRs having antigen specificity for mutant human p53.The two amino acid sequences in each collection correspond to the α and β chains of the TCR, respectively.

[0026] 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.

[0027] A functional variant may, for example, comprise the amino acid sequence of a parent TCR, polypeptide, or protein having at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having particular physical and / or chemical properties is replaced with another amino acid having the same chemical or physical properties. For example, a conservative amino acid substitution can 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.

[0028] Alternatively or additionally, the functional variant may comprise the amino acid sequence of the parent TCR, polypeptide or protein with at least one non-conservative amino acid substitution, where the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant, preferably the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide or protein.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The polypeptide may comprise a functional portion of either or both of the alpha and beta chains of a TCR of the invention, for example a functional portion comprising one or more of the CDR1, CDR2, and CDR3 of the variable region(s) of the alpha and / or beta chain of a TCR of the invention. In an embodiment of the invention, the polypeptide is selected from the group consisting of: (1) all of SEQ ID NOs: 27 to 32; (2) all of SEQ ID NOs: 37 to 42; (3) all of SEQ ID NOs: 47 to 52; (4) all of SEQ ID NOs: 57 to 62; (5) all of SEQ ID NOs: 67 to 72; (6) all of SEQ ID NOs: 77 to 82; (7) all of SEQ ID NOs: 87 to 92; (8) all of SEQ ID NOs: 97 to 102; (9) all of SEQ ID NOs: 107 to 112; (10) all of SEQ ID NOs: 117 to 122; (11) all of SEQ ID NOs: 127 to 132; (12) all of SEQ ID NOs: 137 to 142; (13) all of SEQ ID NOs: 147 to 152; (14) all of SEQ ID NOs: 157 to 162; (15) all of SEQ ID NOs: 167 to 172; (16) SEQ ID NO: 17 (17) all of SEQ ID NOs: 187 to 192; (18) all of SEQ ID NOs: 197 to 202; (19) all of SEQ ID NOs: 207 to 212; (20) all of SEQ ID NOs: 217 to 222; (21) all of SEQ ID NOs: 227 to 232; (22) all of SEQ ID NOs: 237 to 242; (23) all of SEQ ID NOs: 247 to 252; (24) all of SEQ ID NOs: 257 to 262 (25) all of SEQ ID NOs: 267 to 272; (26) all of SEQ ID NOs: 277 to 282; (27) all of SEQ ID NOs: 287 to 292; (28) all of SEQ ID NOs: 297 to 302; (29) all of SEQ ID NOs: 307 to 312; (30) all of SEQ ID NOs: 317 to 322; (31) all of SEQ ID NOs: 327 to 332; (32) all of SEQ ID NOs: 337 to 342 (33) all of SEQ ID NOs: 347 to 352; (34) all of SEQ ID NOs: 357 to 362; (35) all of SEQ ID NOs: 367 to 372; (36) all of SEQ ID NOs: 377 to 382; (37) all of SEQ ID NOs: 387 to 392; (38) all of SEQ ID NOs: 397 to 402; (39) all of SEQ ID NOs: 407 to 412; (40) all of SEQ ID NOs: 417 to 422; (41) all of SEQ ID NOs: 427 to 432; (42) all of SEQ ID NOs: 437 to 442; (43) all of SEQ ID NOs: 447 to 452; (44) all of SEQ ID NOs: 457 to 462; (45) all of SEQ ID NOs: 467 to 472; (46) all of SEQ ID NOs: 477 to 482; or (47) all of SEQ ID NOs: 487 to 492.

[0034] 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 CDR regions. In this regard, the polypeptide may comprise: (1) both of SEQ ID NOs: 33 to 34; (2) both of SEQ ID NOs: 43 to 44; (3) both of SEQ ID NOs: 53 to 54; (4) both of SEQ ID NOs: 63 to 64; (5) both of SEQ ID NOs: 73 to 74; (6) both of SEQ ID NOs: 83 to 84; (7) both of SEQ ID NOs: 93 to 94; (8) both of SEQ ID NOs: 103 to 104; (9) both of SEQ ID NOs: 113 to 114; (10) both of SEQ ID NOs: 123 to 124; (11) both of SEQ ID NOs: 133 to 134; (12) both of SEQ ID NOs: 143 to 144; (13) (14) both of SEQ ID NOs: 163 to 164; (15) both of SEQ ID NOs: 173 to 174; (16) both of SEQ ID NOs: 183 to 184; (17) both of SEQ ID NOs: 193 to 194; (18) both of SEQ ID NOs: 203 to 204; (19) both of SEQ ID NOs: 213 to 214; (20) both of SEQ ID NOs: 223 to 224; (21) both of SEQ ID NOs: 233 to 234; (22) both of SEQ ID NOs: 243 to 244; (23) both of SEQ ID NOs: 253 to 254; (24) both of SEQ ID NOs: 263 to 264; ( (25) both of SEQ ID NOs: 273 to 274; (26) both of SEQ ID NOs: 283 to 284; (27) both of SEQ ID NOs: 293 to 294; (28) both of SEQ ID NOs: 303 to 304; (29) both of SEQ ID NOs: 313 to 314; (30) both of SEQ ID NOs: 323 to 324; (31) both of SEQ ID NOs: 333 to 334; (32) both of SEQ ID NOs: 343 to 344; (33) both of SEQ ID NOs: 353 to 354; (34) both of SEQ ID NOs: 363 to 364; (35) both of SEQ ID NOs: 373 to 374; (36) both of SEQ ID NOs: 383 to 384 (37) both of SEQ ID NOs: 393 to 394; (38) both of SEQ ID NOs: 403 to 404; (39) both of SEQ ID NOs: 413 to 414; (40) both of SEQ ID NOs: 423 to 424; (41) both of SEQ ID NOs: 433 to 434; (42) both of SEQ ID NOs: 443 to 444; (43) both of SEQ ID NOs: 453 to 454; (44) both of SEQ ID NOs: 463 to 464; (45) both of SEQ ID NOs: 473 to 474; (46) both of SEQ ID NOs: 483 to 484; or (47) both of SEQ ID NOs: 493 to 494.

[0035] 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 the amino acid sequence of (i) one of SEQ ID NOs: 23 to 25 or (ii) one of SEQ ID NOs: 25 and 23 to 24.

[0036] In an embodiment of the present invention, the polypeptide of the present invention may comprise the α chain and the β chain of the TCR of the present invention. In this regard, the polypeptide may comprise: (1) both of SEQ ID NOs: 35 to 36; (3) both sequence numbers 55-56; (4) both sequence numbers 65-66; (5) both sequence numbers 75-76; (6) both sequence numbers 85-86; (7) both sequence numbers 95-96; (8) both sequence numbers 105-106; (9) both sequence numbers 115-116; (10) both sequence numbers 125-126; (11) both sequence numbers 135-136; (12) both sequence numbers 145-146; (13) both sequence numbers 155-156; (14) both sequence numbers 165- (15) both of SEQ ID NOs: 175-176; (16) both of SEQ ID NOs: 185-186; (17) both of SEQ ID NOs: 195-196; (18) both of SEQ ID NOs: 205-206; (19) both of SEQ ID NOs: 215-216; (20) both of SEQ ID NOs: 225-226; (21) both of SEQ ID NOs: 235-236; (22) both of SEQ ID NOs: 245-246; (23) both of SEQ ID NOs: 255-256; (24) both of SEQ ID NOs: 265-266; (25) both of SEQ ID NOs: 275-276; (26) both of SEQ ID NOs: 285 to 286; (27) both of SEQ ID NOs: 295 to 296; (28) both of SEQ ID NOs: 305 to 306; (29) both of SEQ ID NOs: 315 to 316; (30) both of SEQ ID NOs: 325 to 326; (31) both of SEQ ID NOs: 335 to 336; (32) both of SEQ ID NOs: 345 to 346; (33) both of SEQ ID NOs: 355 to 356; (34) both of SEQ ID NOs: 365 to 366; (35) both of SEQ ID NOs: 375 to 376; (36) both of SEQ ID NOs: 385 to 386; (37) both of SEQ ID NOs: (39) both of SEQ ID NOs: 395 to 396; (38) both of SEQ ID NOs: 405 to 406; (39) both of SEQ ID NOs: 415 to 416; (40) both of SEQ ID NOs: 425 to 426; (41) both of SEQ ID NOs: 435 to 436; (42) both of SEQ ID NOs: 445 to 446; (43) both of SEQ ID NOs: 455 to 456; (44) both of SEQ ID NOs: 465 to 466; (45) both of SEQ ID NOs: 475 to 476; (46) both of SEQ ID NOs: 485 to 486; or (47) both of SEQ ID NOs: 495 to 496.

[0037] 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: 27 to 29 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 30 to 32; (2) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 37 to 39 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 40 to 42; (3) 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; (4) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 57 to 59 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: (5) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 67 to 69 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 70 to 72; (6) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 77 to 79 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 80 to 82; (7) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 87 to 89 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 90 to 92. Polypeptide chain; (8) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 97 to 99 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 100 to 102; (9) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 107 to 109 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 110 to 112; (10) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 117 to 119 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 120 to 122; (11) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 127 to 129 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: (12) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 137 to 139 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 140 to 142; (13) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 147 to 149 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 150 to 152; (14) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 157 to 159 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 1.(15) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 167 to 169 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 170 to 172; (16) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 177 to 179 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 180 to 182; (17) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 187 to 189 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 190 to 192. (18) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 197 to 199 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 200 to 202; (19) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 207 to 209 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 210 to 212; (20) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 217 to 219 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 220 to 222. (21) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 227 to 229 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 230 to 232; (22) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 237 to 239 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 240 to 242; (23) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 247 to 249 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 250 to 252. Polypeptide chain; (24) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 257 to 259 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 260 to 262; (25) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 267 to 269 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 270 to 272; (26) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 277 to 279 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 280 to 282;(27) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 287 to 289 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 290 to 292; (28) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 297 to 299 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 300 to 302; (29) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 307 to 309 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 310 to 312; (30) all of SEQ ID NOs: 317 to 319 (31) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 327 to 329 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 330 to 332; (32) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 337 to 339 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 340 to 342; (33) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 347 to 349. (34) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 357 to 359 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 360 to 362; (35) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 367 to 369 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 370 to 372; (36) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 377 to 379 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 380 to 382. (37) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 387 to 389 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 390 to 392; (38) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 397 to 399 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 400 to 402; (39) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 407 to 409 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 410 to 412;(40) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 417 to 419 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 420 to 422; (41) all of the amino acids of SEQ ID NOs: 427 to 429; (41) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 430 to 432 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 437 to 439 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 440 to 442; (43) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 447 to 449 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 450 to 452; (44) a first polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 457 to 459 and a second polypeptide chain including all of the amino acid sequences of SEQ ID NOs: 460 to 462 (45) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 467 to 469 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 470 to 472; (46) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 477 to 479 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 480 to 482; or (47) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 487 to 489 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 490 to 492.

[0038] In an embodiment of the invention, the protein comprises: (1) 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; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 43 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 44; (3) 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; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 63 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 64; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 73 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 74; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 84; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 93 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 94; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 103 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 104; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 113 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 114; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 123 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 124; (11) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 133 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 134; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 143 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 144; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 154; (14) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 163 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 164; (15) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 173 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 174; (16) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 183 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 184;(17) 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: 194; (18) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 203 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 204; (19) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 213 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 214; (20) 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: 224. (21) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:233 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:234; (22) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:243 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:244; (23) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:253 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:254; (24) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:263 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:264; (25) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 273 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 274; (26) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 283 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 284; (27) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 293 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 294; (28) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 303 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 304. (29) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 313 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 314; (30) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 323 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 324; (31) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 333 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 334; (32) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 343 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 344. (33) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 353 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 354; (34) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 363 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 364; (35) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 373 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 374; (36) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 383 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 384. Peptide chains; (37) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 393 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 394; (38) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 403 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 404; (39) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 413 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 414; (40) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 423 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 424;(41) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 433 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 434; (42) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 443 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 444; (43) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 453 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 454; (44) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 463 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 464; (45) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 473 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 474; (46) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 483 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 484; or (47) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 493 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 494.

[0039] In an embodiment of the invention, the protein comprises: (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:35 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:36; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:45 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:46; (3) 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; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:65 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:7 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:7. (6) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 85 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 86; (7) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 95 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 96; (8) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 105 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 106; (9) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO: 115 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO: 120.(10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 125 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 126; (11) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 135 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 136; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 145 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 146; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 155. (14) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 166; (15) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 175 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 176; (16) 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: 186; (17) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 195 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 196; (18) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 205 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 206; (19) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 215 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 216; (20) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 225 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226; (21) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 225 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226; (22) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:245 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:246; (23) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:255 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:256; (24) a first polypeptide chain comprising an amino acid sequence of SEQ ID NO:265 and a second polypeptide chain comprising an amino acid sequence of SEQ ID NO:266;(25) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:275 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:276; (26) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:285 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:286; (27) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:295 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:296; (28) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:305 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:306; (29) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 315 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 316; (30) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 325 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 326; (31) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 335 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 336; (32) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 345 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 346; (33) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 355 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 356; (34) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 365 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 366; (35) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 375 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 376; (36) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 385 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 386; (37) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 395 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 396; (38) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 405 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 406; (39) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 415 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 416; (40) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 425 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 426;(41) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 435 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 436; (42) a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 445; (43) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 455 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 456; (44) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 465 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 466; (45) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 475 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 476; (46) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 485 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 486; or (47) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 495 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 496.

[0040] 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 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 immunoglobulins, CD3, CD4, CD8, MHC molecules, CD1 molecules, such as CD1a, CD1b, CD1c, CD1d, etc.

[0041] 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 include, for example, recombinant methods.

[0042] 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 alpha and beta 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: 26. Once the construct comprising the linker peptide is expressed by the host cell, the linker peptide may be cleaved, resulting in separated alpha and beta chains. In embodiments of the invention, the TCRs, polypeptides, or proteins may comprise an amino acid sequence comprising a full-length alpha chain, a full-length beta chain, and a linker peptide located between the alpha and beta chains.

[0043] 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 the antibody heavy chain, light chain, heavy or light chain variable or constant region, single chain variable region (scFv), or Fc, Fab, or F(ab)2' fragment. The antibody polypeptide chain or antigen-binding portion thereof may be present as separate polypeptides of a recombinant antibody. Alternatively, the antibody polypeptide chain or antigen-binding portion thereof may be present as a polypeptide expressed in frame (tandem) with a polypeptide of the invention. The antibody polypeptide or antigen-binding portion thereof The molecule may be any antibody or any antibody fragment polypeptide, including any of the antibodies and antibody fragments described herein.

[0044] 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, such as 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.

[0045] The TCRs, polypeptides and proteins of the invention may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline, 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.

[0046] 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.

[0047] 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.

[0048] An embodiment of the present invention provides a nucleic acid comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. "Nucleic acid," as used herein, includes "polynucleotides," "oligonucleotides," and "nucleic acid molecules." It 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 bonds, such as phosphoramidate or phosphorothioate bonds, instead of the phosphodiesters found between nucleotides of unmodified oligonucleotides. In embodiments, nucleic acids include complementary DNA (cDNA). It is generally preferred that nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, it may be preferred that nucleic acids contain one or more insertions, deletions, inversions, and / or substitutions, as discussed herein.

[0049] 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.

[0050] Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. See, e.g., Green and Sambrook, 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-galactosylqueosine, inosine, N 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -Substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6-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).

[0051] 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.

[0052] 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.

[0053] 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 the nucleic acids described herein) in an amount detectably greater than non-specific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences or those containing only a few scattered mismatches from random sequences that happen to have a few small regions (e.g., 3-10 bases) 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 tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the TCRs of the present invention. It will generally be appreciated that conditions can be made more stringent by adding increasing amounts of formamide.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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 vectors for propagation and propagation, or vectors for expression, such as plasmids and viruses. Examples of expression vectors include those designed for plant, plant, or both. Vectors 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-based vector or a viral vector, such as a retroviral vector.

[0058] 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. Expression vector constructs, which may be circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, lambda, SV40, bovine papilloma virus, etc.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] Additionally, recombinant expression vectors may be engineered to contain a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell in which it is expressed to die. A suicide gene may provide protection against an agent, e.g., a drug, to the cell in which it is expressed. The suicide gene may be a gene that confers sensitivity to an agent and causes the cell to die when the cell is 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.

[0064] 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 or primary cell, i.e., a cell directly isolated from an organism, such as a human. The host cell may be an adherent or suspension cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For purposes of producing a recombinant TCR, polypeptide, or protein, the host cell is preferably a mammalian cell. Most preferably, the host cell is a human cell. The host cell may be of any cell type, 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.

[0065] 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.

[0066] 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 a cell other than a T cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, a brain cell, 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.

[0067] In embodiments of the invention, the number of cells in a population can be rapidly expanded, as described, for example, in U.S. Patent Nos. 8,034,334, 8,383,099, U.S. Patent Application Publication No. 2012 / 0244133, Dudley et al., J. Immunother., 26:332-42 (2003), and Riddell et al., J. Immunol. Methods, 128:189-201 (1990). As noted above, expansion of T cell numbers can be accomplished by any of a number of methods known in the art. In embodiments, 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).

[0068] 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%.

[0069] 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 materials of the invention") may be formulated into compositions, such as pharmaceutical compositions. In this regard, the invention provides pharmaceutical compositions comprising any of the TCRs, polypeptides, proteins, nucleic acids, expression vectors, and host cells (including populations thereof) described herein and a pharma- ceutically acceptable carrier. Pharmaceutical compositions of the invention containing any of the TCR materials of the invention may include more than one TCR material of the invention, e.g., a polypeptide and a nucleic acid, or two or more different TCRs. Alternatively, the pharmaceutical composition may include the TCR material of the invention in combination with another pharma- ceutical active agent(s) or drug(s), e.g., a chemotherapeutic agent such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like.

[0070] 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.

[0071] 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.

[0072] 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, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In an embodiment, the pharma- ceutically acceptable carrier is supplemented with human serum albumen.

[0073] 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 an appropriate 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) being treated.

[0074] Many assays for determining the dose to be administered are known in the art. For the purposes of the present invention, an assay can be used to determine the starting dose to be administered to a mammal, which 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 extent to which target cells are lysed or IFN-γ is secreted when a particular dose is administered can be assayed by methods known in the art.

[0075] 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 dose 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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 will recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any amount or level of 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" can include delaying the onset of cancer or a symptom or condition thereof. Alternatively or additionally, "prevention" can include preventing or delaying the recurrence of cancer or a symptom or condition thereof.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In the context of the methods of the present invention, the cancer may be any of 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 tumor, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelial cancer, The cancer may be any cancer, including any of the following: ovarian cancer, 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 mutant p53. The cancer may express p53 having a mutation at any one or more of positions 175, 220, 245, 248, 249, 273, and 282 of SEQ ID NO: 1. The cancer may express p53 with any one or more of the following human p53 mutations: R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273H, R273C, R273L, and R282W. 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.

[0086] The mammal referred to in the method of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including but not limited to rodent mammals, such as mice and hamsters, and lagomorph mammals, such as rabbits. Preferably, the mammal is a mammal of the order Carnivora, including cats and dogs. More preferably, the mammal is a mammal of the order Artiodactyla, including cats and pigs, or Perissodactyla, including horses. Most preferably, the mammal is a mammal of the order Primates, Ceboids or Simoids (monkeys), or Anthropoids (humans and apes). A particularly preferred mammal is a human. EXAMPLES

[0087] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0088] The amino acid sequences listed in Tables 1-3 were used in the experiments described in the following Examples. In Tables 1-2, "LP" means "long peptide." In Table 3, "TMG" means "tandem minigene."

[0089] [Table 1]

[0090] The WT versions of the peptides in Table 1 are listed in Table 2.

[0091] [Table 2]

[0092] [Table 3]

[0093] Example 1 This example shows the isolation and specific reactivity of four anti-mutated p53 TCRs from patient 4127.

[0094] For patient 4127, experiments were performed as described in Figures 1-7, 36, and 37A-37C. Mutant p53-reactive T cells from this patient were identified by the method described in US Patent Application Publication No. 2017 / 0224800 (the "Tran Method"). The method for isolating individual TCRs is described below.

[0095] Autologous PBL were transduced with one of the TCRs shown in Figure 1 and tested 2 weeks later. Autologous DC cells were transduced at 3 × 10 4 Cells / well were plated and pulsed overnight with decreasing peptide concentrations of either the WT p53-G245 peptide (wt) or the mutant p53-G245S peptide (mut). 3 × 10 cells / well were plated and pulsed overnight with decreasing peptide concentrations of either the WT p53-G245 peptide (wt) or the mutant p53-G245S peptide (mut). 4 Transduced T cells were added and co-cultured overnight at 37°C. Supernatants were collected for IFN-γ ELISA. IFN-γ ELISA results are shown in Figure 1. FACS gates: Lymphocytes\PI(negative)CD3+ \CD3 + mTCR + \CD8(negative)CD4 + The FACS results are shown in FIG.

[0096] Autologous PBL were transduced with one of the TCRs shown in FIG. 3 and were treated with either DMSO (peptide vehicle) or the 15mer p53-G245S peptide with a 14 amino acid overlap shown in Table A. Co-culture was performed as described for the experiment in Figure 2, except that DCs were pulsed with one of the peptides. 4-1BB expression was performed as described in Figure 2. The results are shown in Figure 3.

[0097] [Table A]

[0098] Cos7 cells (2.5×10 4 T cells (10 cells / well) were plated into wells of a flat-bottom 96-well plate. After 20 hours, cells were co-transfected with individual HLA alleles. After 20 hours, cells were pulsed with 10 μg / mL of p53G245S-15mer peptide for 3 hours at 37°C. After washing, T cells (10 5 ) were added to the wells and co-cultured overnight (20 h) at 37°C. IFN-γ secretion was measured by ELISA; predicted by NetMHCIIpan: cbs.dtu.dk / services / NetMHCIIpan / . 4-1BB expression was measured by FACS. FACS gate: lymphocytes → live cells (PI negative) → CD3+ (T cells) → CD4+ (4127-TIL) or CD4+mTCR+ (TCR-transduced T cells). The results are shown in Figures 4-5 and Table B.

[0099] [Table B]

[0100] DRB3 *DRB3 is expressed in 1367 of 3719 (37%) DRB-type patients in the NCI HLA database and in 5 of 9 (56%) endometrial and ovarian cancer patients in the NCI Surgery Branch. * The frequency of the 02 allele is very high according to the allelefrequencies.net website. For example, this website states that the DRB3 * The frequency of the 02 allele is USA NMDP Middle Eastern or North Coast of Africa It has been reported that the population size is 0.3447.

[0101] TILs from patient 4127 were either (1) electroporated with tandem minigenes (TMG) composed of irrelevant, WT p53, or mutant p53 sequences, or (2) pulsed with peptide vehicle (DMSO) or purified (>95% by high performance liquid chromatography (HPLC)) 25 amino acid peptides composed of the WT p53-G245 or mutant p53-G245S sequences, allogeneic (DRB3 * 01:01:01 or DRB3 * 02:02:01) Co-cultured with antigen-presenting cells (APCs). was carried out overnight at 37° C. IFN-γ secretion was assessed using an ELISPOT assay, and the results are shown in FIG.

[0102] T cells expressing 4127-TCR1 specific for p53-G245S were co-cultured with allogeneic APCs as described for the experiment in Figure 6. The results are shown in Figure 7.

[0103] Autologous APCs were pulsed with decreasing concentrations of a 25 amino acid peptide corresponding to the sequence of WTp53-G245 or mutant p53-G245S. 4127-O37-TCR-transduced T cells from patient 4127 were co-cultured with peptide-pulsed APCs overnight at 37°C. After gating on lymphocytes → single cells → live cells → CD3+mTCR+, 4-1BB expression was assayed by flow cytometry. The results are shown in Figure 36.

[0104] Cos7 cells (2.5×10 4 10 cells / well) were plated into wells of a flat-bottom 96-well plate. The following day, individual HLA alleles from patient 4127 were co-transfected into the cells. The following day, the 25 amino acid p53-G245S peptide was pulsed onto the transfected Cos7 cells. Excess peptide was washed away. T cells were transduced with one of the TCRs from patient 4127. The transduced T cells were added to the co-culture with Cos7 cells (2 × 10 4 Co-cultures were incubated overnight at 37°C. IFN-γ secretion was assessed by ELISA. 4-1BB expression was assayed by flow cytometry after gating on lymphocytes → single cells → live cells → CD3+mTCR+. Results are shown in Figures 37A-37C.

[0105] The sequence of TCR4127-TP53-G245S-TCR1 isolated from patient 4127 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:30), the second underlined region is CDR2 beta (SEQ ID NO:31), the third underlined region is CDR3 beta (SEQ ID NO:32), the fourth underlined region is CDR1 alpha (SEQ ID NO:27), the fifth underlined region is CDR2 alpha (SEQ ID NO:28), and the sixth underlined region is CDR3 alpha (SEQ ID NO:29). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:25), and the second italicized region is the alpha chain constant region (SEQ ID NO:23). The beta chain variable region (SEQ ID NO:34) includes the sequence beginning at the amino terminus and ending just before the beginning of the beta chain constant region. The alpha chain variable region (SEQ ID NO:33) contains the sequence beginning immediately after the linker and ending just before the beginning of the alpha chain constant region. The full-length beta chain (SEQ ID NO:36) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length alpha chain (SEQ ID NO:35) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0106] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR Name: 4127-TP53-G245S-TCR1 Recognizing p53 mutation: G245S Method: Tran method Co-culture to identify TCR: Co-culture 4127-F10 TIL fragments with G245S long peptide and sort CD4+41BB+ T cells (single cell RT-PCR only) Methods for identifying TCR: Single-cell RT-PCR (below), Adaptive Pairseq (4127-F10 fragment), 5'RACE from TIL clone O71 TCR abundance among all paired TCRs: 31.3% (5 observations out of 16 pairs) TCR orientation: beta-alpha Expression vector: gamma-retrovirus

[0107] [ka]

[0108] Statistics for TCR4127-TP53-G245S-TCR1 for patient 4127 are listed in Table 4 below.

[0109] [Table 4]

[0110] The sequence of TCR4127-TP53-G245S-TCR4 isolated from patient 4127 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:40), the second underlined region is CDR2 beta (SEQ ID NO:41), the third underlined region is CDR3 beta (SEQ ID NO:42), the fourth underlined region is CDR1 alpha (SEQ ID NO:37), the fifth underlined region is CDR2 alpha (SEQ ID NO:38), and the sixth underlined region is CDR3 alpha (SEQ ID NO:39). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:25), and the second italicized region is the alpha chain constant region (SEQ ID NO:23). The beta chain variable region (SEQ ID NO:44) includes the sequence beginning at the amino terminus and ending just before the beginning of the beta chain constant region. The alpha chain variable region (SEQ ID NO:43) contains the sequence beginning immediately after the linker and ending just before the beginning of the alpha chain constant region. The full length beta chain (SEQ ID NO:46) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full length alpha chain (SEQ ID NO:45) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0111] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR Name: 4127-TP53-G245S-TCR4 Recognizing p53 mutation: G245S Method: Tran method Co-culture to identify TCR: Co-culture 4127-F10 TIL fragments with G245S long peptide and sort CD4+41BB+ T cells (single cell RT-PCR only) Methods for identifying TCR: Single-cell RT-PCR (below) and Adaptive Pairseq (4127-F10 fragment) TCR abundance among all paired TCRs: 12.5% ​​(observed 2 times out of 16 pairs) TCR orientation: beta-alpha Expression vector: gamma-retrovirus

[0112] [ka]

[0113] Statistics for TCR4127-TP53-G245S-TCR4 for patient 4127 are listed in Table 5 below.

[0114] [Table 5]

[0115] The sequence of TCR4127_O102_TCR isolated from patient 4127 is set forth below. 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:26). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:25), and the second italicized region is the alpha chain constant region (SEQ ID NO:23). The beta chain variable region (SEQ ID NO:54) includes the sequence beginning at the amino terminus and ending just before the beginning 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 beginning 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 beginning 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.

[0116] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR name: 4127_O102_TCR Recognizing p53 mutation: G245S Method: Tran method Co-culture to identify TCR: Not performed. TCR was identified directly from the RNA of T cell clone (0102) using 5'RACE. Clones were developed by sorting for OX40+ T cells and establishing T cell clones by limiting dilution. T cell clones were screened using the Tran method. Method for identifying TCR: 5'RACE from TIL clone O102 TCR abundance among all paired TCRs: 100% of T cell clones (not applicable) TCR orientation: beta-alpha Expression vector: gamma-retrovirus

[0117] [ka]

[0118] The sequence of TCR4127-O37-TCR isolated from patient 4127 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:460), the second underlined region is CDR2 beta (SEQ ID NO:461), the third underlined region is CDR3 beta (SEQ ID NO:462), the fourth underlined region is CDR1 alpha (SEQ ID NO:457), the fifth underlined region is CDR2 alpha (SEQ ID NO:458), and the sixth underlined region is CDR3 alpha (SEQ ID NO:459).

[0119] The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:25) and the second italicized region is the alpha chain constant region (SEQ ID NO:23). The beta chain variable region (SEQ ID NO:464) contains the sequence starting at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:463) contains the sequence starting just after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:466) contains the sequence starting at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:465) contains the sequence starting just after the linker and ending at the carboxyl terminus.

[0120] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR name: 4127-O37-TCR Recognizing p53 mutation: G245S Method: Tran method Co-culture to identify TCR: Not performed. TCR was identified directly from the genomic DNA of T cell clone (037) using Adaptive TCRAD and TCRB Survey (Next Generation Sequencing (NGS) platform). O37 clone was developed by sorting OX40+ T cells and establishing T cell clones by limiting dilution. T cell clones were screened using the Tran method. Method for identifying TCR: Adaptive TCR sequencing (NGS) of TIL clone O37 TCR abundance among all paired TCRs: 100% of T cell clones (n / a) TCR orientation: beta-alpha Expression vector: gamma-retrovirus

[0121] [ka]

[0122] Example 2 This example shows the isolation and specific reactivity of three anti-mutated p53 TCRs from patient 4196.

[0123] For patient 4196, experiments were performed as described in Figures 8-11. p53-reactive T cells from this patient were identified by the Tran method described in US Patent Application Publication No. 2017 / 0224800. The Fluidigm method was used to isolate individual TCRs. Statistics for patient 4196 are shown in Table D.

[0124] Characterization of p53-responsive cells: Attempts to identify the minimal epitope: mutant amino acid p53 The initially predicted peptide with R175H is shown in Table C.

[0125] [Table C]

[0126] TMG1 vs. 4196-Rx1 TIL: Plasmids for TMG1 and HLA alleles were transduced into Cos7 cells. Cells were co-cultured with 4196 Rx1 TIL infusion bags for 20 hours (h). Results are shown in Figure 8A.

[0127] P53-R175H minimal peptide versus 4196-Rx1 TIL: On day 4, autologous DCs were pulsed with either the candidate minimal epitopes (HMTEVVRHC (SEQ ID NO: 530) or (SQHMTEVVRH (SEQ ID NO: 531)) for 2 hours. Cells were washed and co-cultured with 4196 Rx1 TIL infusion bags for 20 hours. Results are shown in FIG. 8B.

[0128] A * 02:01-restrictive TP53 tetramers can be used to isolate TP53-reactive cells from TIL infusion bags: Isolation of TP53-reactive cells using 4-1BB+ sorting after co-culture with TP53 peptide was technically limited. To isolate TP53-reactive cells, A * 02:01-TP53 tetramer was generated. * 02:01-gp100) was generated as a control. FACS analysis was performed by gating on live cells, CD3+ cells, and CD8+ cells. * For the 02:01-TP53 tetramer, 14.9% of p53 tetramer+ cells were detected. * For the 02:01-gp100 tetramer, 0.7% of p53 tetramer+ cells were detected.

[0129] Isolation and characterization of TP53-reactive cells: The strategy for isolating and characterizing TP53-reactive cells was as follows: (1) sort tetramer-positive cells using FACS as described above, (2) perform single-cell PCR, and (3) perform Vβ seep sequencing.

[0130] Using single cell PCR, several candidate clones were identified for the TP53 TCR, as shown in Table D. TCR-1a and TCR-1b were further candidate clones.

[0131] [Table D]

[0132] Plasmids encoding candidate TCRs were cloned into the MSGV1 vector, and the TCRs were introduced into donor lymphocytes using retroviral transduction.

[0133] On the fourth day, A * 02:01 DCs were pulsed with HPLC-grade minimal epitopes for 2 hours. DCs were co-cultured with TCR-transduced cells for 20 hours. The results are shown in Figures 9A-9D. * 02:01 Restrictive TP53 receptor identified.

[0134] T2 cells were thawed, left to rest for 24 hours, then pulsed with decreasing concentrations of HPLC minimal epitopes for 2 hours. Cells were then washed and co-cultured with TP53 TCR transduced cells for 17 hours. The results are shown in Figures 10A-10B.

[0135] The following parental tumor cell lines were identified: * 02:01 was transduced with: colon line - LS123 (ATCC CCL-255) with R175H but unknown HLA-A2 status; leukemia line: CCRF-CEM (ATCC CRM-CCL-119, leukemia) with R175H but unknown HLA-A2 status; breast line: AU-565 (ATCC CRL-2351, breast adenocarcinoma) with R175H but unknown HLA-A2 status; melanoma cell line: MEL624; endogenous expression of HLA-A2 and wt R175.

[0136] Target-transduced tumor cells were harvested and plated the morning of coculture (1 × 10 5R175H minimal epitope was pulsed onto target cells (HPLC, 5 μg / mL) for 2 h. Cells were washed twice and 2 × 10 4 TP53 TCR transduced cells for 20 hours (Patient 4196). IFN-γ ELISPOT was used to read out the response. Results are shown in Figure 11. Further experimental data regarding target cell HLA-A2 and TP53 expression and TCR reactivity in this example are provided in Example 15.

[0137] The sequence of TCR4196_AV12-1_with_BV6-1 isolated from patient 4196 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO: 70), the second underlined region is CDR2 beta (SEQ ID NO: 71), the third underlined region is CDR3 beta (SEQ ID NO: 72), the fourth underlined region is CDR1 alpha (SEQ ID NO: 67), the fifth underlined region is CDR2 alpha (SEQ ID NO: 68), and the sixth underlined region is CDR3 alpha (SEQ ID NO: 69). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the beta The first italicized region is the data chain constant region (SEQ ID NO:25) and the second italicized region is the alpha chain constant region (SEQ ID NO:24). The beta chain variable region (SEQ ID NO:74) contains the sequence starting at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:73) contains the sequence starting just after the linker and ending just before the start of the alpha chain constant region. The full length beta chain (SEQ ID NO:76) contains the sequence starting at the amino terminus and ending just before the start of the linker. The full length alpha chain (SEQ ID NO:75) contains the sequence starting just after the linker and ending at the carboxyl terminus.

[0138] Cancer-reactive T cells were identified as follows. TCR name: 4196_AV12-1_with_BV6-1 Recognizing p53 mutation: R175H Method: Tran method

[0139] [ka]

[0140] The sequence of TCR4196_AV38-1_with_BV10-3 isolated from patient 4196 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO:80), the second underlined region is CDR2 beta (SEQ ID NO:81), the third underlined region is CDR3 beta (SEQ ID NO:82), the fourth underlined region is CDR1 alpha (SEQ ID NO:77), the fifth underlined region is CDR2 alpha (SEQ ID NO:78), and the sixth underlined region is CDR3 alpha (SEQ ID NO:79). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO:25), and the second italicized region is the alpha chain constant region (SEQ ID NO:24). The beta chain variable region (SEQ ID NO:84) includes the sequence beginning at the amino terminus and ending just before the beginning of the beta chain constant region. The alpha chain variable region (SEQ ID NO:83) contains the sequence beginning immediately after the linker and ending just before the beginning of the alpha chain constant region. The full length beta chain (SEQ ID NO:86) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full length alpha chain (SEQ ID NO:85) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0141] Cancer-reactive T cells were identified as follows. TCR name: 4196_AV38-1_with_BV10-3 Recognizing p53 mutation: R175H Method: Tran method

[0142] [ka]

[0143] The sequence of TCR4196_AV6_with_BV11-2 isolated from patient 4196 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 beta (SEQ ID NO: 90), the second underlined region is CDR2 beta (SEQ ID NO: 91), the third underlined region is CDR3 beta (SEQ ID NO: 92), the fourth underlined region is CDR1 alpha (SEQ ID NO: 87), the fifth underlined region is CDR2 alpha (SEQ ID NO: 88), and the sixth underlined region is CDR3 alpha (SEQ ID NO: 89). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the beta chain constant region (SEQ ID NO: 25), and the second italicized region is the alpha chain constant region (SEQ ID NO: 24). The beta chain variable region (SEQ ID NO: 94) includes the sequence beginning at the amino terminus and ending just before the beginning of the beta chain constant region. The alpha chain variable region (SEQ ID NO:93) contains the sequence beginning immediately after the linker and ending just before the beginning of the alpha chain constant region. The full length beta chain (SEQ ID NO:96) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full length alpha chain (SEQ ID NO:95) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0144] Cancer-reactive T cells were identified as follows. TCR name: 4196_AV6_with_BV11-2 Recognizing p53 mutation: R175H Method: Tran method

[0145] [ka]

[0146] Example 3 This example shows the isolation of 11 anti-mutated p53 TCRs from patient 4238.

[0147] For patient 4238, the study was performed as described in Figures 12-15.

[0148] TIL fragments (F1-F24, n=24) from patient 4238 were co-cultured with autologous APCs electroporated with TMG composed of irrelevant or mutant p53 sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 12. Lymphocytes → live cells (PI negative) → CD3+ (T cells) After gating, 4-1BB expression was assessed by flow cytometry, and the results are shown in FIG.

[0149] TIL fragments (F1-F24, n=24) from patient 4238 were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248Q sequence. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 13. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 15.

[0150] The source of TCR after sorting of CD8+41BB+ T cells was the TIL fragments F10, F11, and F17.

[0151] The sequence of TCR4238-F10-TCR1 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:207), the second underlined region is CDR2 alpha (SEQ ID NO:208), the third underlined region is CDR3 alpha (SEQ ID NO:209), the fourth underlined region is CDR1 beta (SEQ ID NO:210), the fifth underlined region is CDR2 beta (SEQ ID NO:211), and the sixth underlined region is CDR3 beta (SEQ ID NO:212). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:213) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:214) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:215) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:216) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0152] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F10-TCR1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F10 with R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell reverse transcriptase polymerase chain reaction (RT-PCR) TCR abundance among all paired TCRs: 25.0% (observed 3 times out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0153] [ka]

[0154] Statistics for TCR4238-F10-TCR1 for patient 4238 are listed in Table 6 below.

[0155] [Table 6]

[0156] The sequence of TCR4238-F10-TCR2 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:217), the second underlined region is CDR2 alpha (SEQ ID NO:218), the third underlined region is CDR3 alpha (SEQ ID NO:219), the fourth underlined region is CDR1 beta (SEQ ID NO:220), the fifth underlined region is CDR2 beta (SEQ ID NO:221), and the sixth underlined region is CDR3 beta (SEQ ID NO:222). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:223) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:224) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:225) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:226) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0157] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F10-TCR2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F10 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 25.0% (observed 3 times out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0158] [ka]

[0159] Statistics for TCR4238-F10-TCR2 for patient 4238 are listed in Table 7 below.

[0160] [Table 7]

[0161] The sequence of TCR4238-F10-TCR3 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:227), the second underlined region is CDR2 alpha (SEQ ID NO:228), the third underlined region is CDR3 alpha (SEQ ID NO:229), the fourth underlined region is CDR1 beta (SEQ ID NO:230), the fifth underlined region is CDR2 beta (SEQ ID NO:231), and the sixth underlined region is CDR3 beta (SEQ ID NO:232). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:233) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:234) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:235) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:236) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0162] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F10-TCR3 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F10 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 58.3% (observed 7 out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0163] [ka]

[0164] Statistics for TCR4238-F10-TCR3 for patient 4238 are listed in Table 8 below.

[0165] [Table 8]

[0166] The sequence of TCR4238-F10-TCR4 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:237), the second underlined region is CDR2 alpha (SEQ ID NO:238), the third underlined region is CDR3 alpha (SEQ ID NO:239), the fourth underlined region is CDR1 beta (SEQ ID NO:240), the fifth underlined region is CDR2 beta (SEQ ID NO:241), and the sixth underlined region is CDR3 beta (SEQ ID NO:242). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:243) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:244) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:245) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:246) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0167] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F10-TCR4 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F10 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 58.3% (observed 7 out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0168] [ka]

[0169] Statistics for TCR4238-F10-TCR4 for patient 4238 are listed in Table 9 below.

[0170] [Table 9]

[0171] The sequence of TCR4238-F11-TCR1 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:247), the second underlined region is CDR2 alpha (SEQ ID NO:248), the third underlined region is CDR3 alpha (SEQ ID NO:249), the fourth underlined region is CDR1 beta (SEQ ID NO:250), the fifth underlined region is CDR2 beta (SEQ ID NO:251), and the sixth underlined region is CDR3 beta (SEQ ID NO:252). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:253) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:254) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:255) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:256) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0172] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F11-TCR1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F11 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 25.0% (observed 3 times out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0173] [ka]

[0174] Statistics for TCR4238-F11-TCR1 for patient 4238 are listed in Table 10 below.

[0175] [Table 10]

[0176] The sequence of TCR4238-F11-TCR2 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:257), the second underlined region is CDR2 alpha (SEQ ID NO:258), the third underlined region is CDR3 alpha (SEQ ID NO:259), the fourth underlined region is CDR1 beta (SEQ ID NO:260), the fifth underlined region is CDR2 beta (SEQ ID NO:261), and the sixth underlined region is CDR3 beta (SEQ ID NO:262). The bolded region is the linker (SEQ ID NO:262). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:263) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:264) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO:265) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO:266) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0177] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F11-TCR2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F11 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 25.0% (observed 3 times out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0178] [ka]

[0179] Statistics for TCR4238-F11-TCR2 for patient 4238 are listed in Table 11 below.

[0180] [Table 11]

[0181] The sequence of TCR4238-F11-TCR3 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:267), the second underlined region is CDR2 alpha (SEQ ID NO:268), the third underlined region is CDR3 alpha (SEQ ID NO:269), the fourth underlined region is CDR1 beta (SEQ ID NO:270), the fifth underlined region is CDR2 beta (SEQ ID NO:271), and the sixth underlined region is CDR3 beta (SEQ ID NO:272). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:273) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:274) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:275) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:276) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0182] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F11-TCR3 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F11 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 16.7% (2 observations out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0183] [ka]

[0184] Statistics for TCR4238-F11-TCR3 for patient 4238 are listed in Table 12 below.

[0185] [Table 12]

[0186] The sequence of TCR4238-F11-TCR4 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:277), the second underlined region is CDR2 alpha (SEQ ID NO:278), the third underlined region is CDR3 alpha (SEQ ID NO:279), the fourth underlined region is CDR1 beta (SEQ ID NO:280), the fifth underlined region is CDR2 beta (SEQ ID NO:281), and the sixth underlined region is CDR3 beta (SEQ ID NO:282). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:283) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:284) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:285) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:286) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0187] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F11-TCR4 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F11 with R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 16.7% (2 observations out of 12 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0188] [ka]

[0189] Statistics for TCR4238-F11-TCR4 for patient 4238 are listed in Table 13 below.

[0190] [Table 13]

[0191] The sequence of TCR4238-F17-TCR1 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:287), the second underlined region is CDR2 alpha (SEQ ID NO:288), the third underlined region is CDR3 alpha (SEQ ID NO:289), the fourth underlined region is CDR1 beta (SEQ ID NO:290), the fifth underlined region is CDR2 beta (SEQ ID NO:291), and the sixth underlined region is CDR3 beta (SEQ ID NO:292). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:293) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:294) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:295) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:296) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0192] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F17-TCR1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F17 with R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 33.3% (observed 2 out of 6 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0193] [ka]

[0194] Statistics for TCR4238-F17-TCR1 for patient 4238 are listed in Table 14 below.

[0195] [Table 14]

[0196] The sequence of TCR4238-F17-TCR2 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:297), the second underlined region is CDR2 alpha (SEQ ID NO:298), the third underlined region is CDR3 alpha (SEQ ID NO:299), the fourth underlined region is CDR1 beta (SEQ ID NO:300), the fifth underlined region is CDR2 beta (SEQ ID NO:301), and the sixth underlined region is CDR3 beta (SEQ ID NO:302). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:303) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:304) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:305) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:306) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0197] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F17-TCR2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F17 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 33.3% (observed 2 out of 6 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0198] [ka]

[0199] Statistics for TCR4238-F17-TCR2 for patient 4238 are listed in Table 15 below.

[0200] [Table 15]

[0201] The sequence of TCR4238-F17-TCR3 isolated from patient 4238 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:307), the second underlined region is CDR2 alpha (SEQ ID NO:308), the third underlined region is CDR3 alpha (SEQ ID NO:309), the fourth underlined region is CDR1 beta (SEQ ID NO:310), the fifth underlined region is CDR2 beta (SEQ ID NO:311), and the sixth underlined region is CDR3 beta (SEQ ID NO:312). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:313) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:314) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:315) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:316) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0202] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4238-F17-TCR3 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4238-F17 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 33.3% (observed 2 out of 6 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0203] [ka]

[0204] Statistics for TCR4238-F17-TCR3 for patient 4238 are listed in Table 16 below.

[0205] [Table 16]

[0206] Example 4 This example shows the isolation of two anti-mutated p53 TCRs from patient 4253.

[0207] For patient 4253, the study was performed as described in FIG.

[0208] TIL fragments (F1-F24; n=24) from patient 4253 were co-cultured with autologous APCs that were (1) pulsed with peptide vehicle (DMSO), (2) pulsed with purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248W sequence, (3) electroporated with irrelevant TMG, or (4) electroporated with p53-mut-TMG containing the mutant p53-R248W sequence. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. Results are shown in Figure 16. F15 was chosen for TCR sorting because it was reactive to both p53-mut-TMG and the p53-R248W long peptide.

[0209] The sequence of TCR4253-TIL-TCR1 isolated from patient 4253 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:187), the second underlined region is CDR2 alpha (SEQ ID NO:188), the third underlined region is CDR3 alpha (SEQ ID NO:189), the fourth underlined region is CDR1 beta (SEQ ID NO:190), the fifth underlined region is CDR2 beta (SEQ ID NO:191), and the sixth underlined region is CDR3 beta (SEQ ID NO:192). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:193) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 194) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 195) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO: 196) contains the sequence beginning immediately after the linker and ending just before the start of the linker. , including sequences ending at the carboxyl terminus.

[0210] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4253-TIL-TCR1 Recognizing p53 mutation: R248W Methods: A simplified universal p53 "hot spot" mutation screen (only mutant TMG and R248W long peptides were evaluated) Co-culture to identify TCR: Co-culture 4253-F15 with p53-R248W long peptide and sort CD3+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 5.6% (observed 2 times out of 36 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0211] [ka]

[0212] Statistics for TCR4253-TIL-TCR1 for patient 4253 are listed in Table 17 below.

[0213] [Table 17]

[0214] The sequence of TCR4253-TIL-TCR2 isolated from patient 4253 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:197), the second underlined region is CDR2 alpha (SEQ ID NO:198), the third underlined region is CDR3 alpha (SEQ ID NO:199), the fourth underlined region is CDR1 beta (SEQ ID NO:200), the fifth underlined region is CDR2 beta (SEQ ID NO:201), and the sixth underlined region is CDR3 beta (SEQ ID NO:202). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:203) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:204) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:205) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:206) contains the sequence beginning immediately after the linker and ending just before the start of the linker. , including sequences ending at the carboxyl terminus.

[0215] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4253-TIL-TCR2 Recognizing p53 mutation: R248W Methods: A simplified universal p53 "hot spot" mutation screen (only mutant TMG and R248W long peptides were evaluated) Co-culture to identify TCR: Co-culture 4253-F15 with p53-R248W long peptide and sort CD3+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 91.7% (observed 33 out of 36 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0216] [ka]

[0217] Statistics for TCR4253-TIL-TCR2 for patient 4253 are listed in Table 18 below.

[0218] [Table 18]

[0219] Example 5 This example shows the identification of anti-mutant p53 T cells in patient 4273 by co-culturing autologous APCs induced to express mutant p53 in autologous T cells ("p53 hotspot mutation universal screening"). This example also shows the isolation of two anti-mutant p53 TCRs from patient 4273.

[0220] For patient 4273, the experiment was performed as described in Figures 17-20 and 56-60.

[0221] TIL fragments (F1–F24, n=24) from patient 4273 were co-cultured with autologous APCs electroporated with TMG composed of irrelevant, WT p53, or mutant p53 sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 17. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). The results are shown in FIG.

[0222] TIL fragments (F1-F24, n=24) from patient 4273 were co-cultured with autologous APC pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 or mutant p53-R248W sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 19. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 20.

[0223] For patient 4273, F15 was the most reactive fragment, and the response by F15 to LP and TMG was comparable and was mainly due to CD4 T cells. Therefore, 4273-F15 was co-cultured with APCs pulsed with R248W LP, and the following day, CD4+41BB+ T cells were sorted as single cells into wells of a 96-well PCR plate at 1 cell / well. The PCR plate has an RT-PCR solution in each well, which amplifies the alpha and beta CDR3 regions of the TCR in the same solution. The wells of the plate are then split into two 96-well PCR plates, and a second PCR round is performed to amplify either CDR3 alpha or CDR3 beta as separate reactions. The PCR products from each well (total of 192 PCR products mapped to each well - alpha or beta) are sequenced by Sanger sequencing. The nucleotide sequences are entered into IMGT / V-QUEST (imgt.org / IMGT_vquest / vquest?livret=0&Option=humanTcR), IgBlast (ncbi.nlm.nih.gov / igblast / igblast.cgi?CMD=Web&SEARCH_TYPE=TCR&LINK_LOC=igtab) and translated by Expasy (web.expasy.org / translate / ). The variable family is determined and fused to the CDR3 and junctions (J or DJ) from the translated sequence. The variable sequences are fused to mouse constant sequences and the rearranged TCR alpha and TCR beta are linked by furin-flexible-P2A (RAKR-SGSG-ATNFSLLKQAGDVEENPGP) (SEQ ID NO: 26). DNA is then synthesized de novo from the sequence and cloned into an expression vector (gamma-retrovirus or SLEEPING BEAUTY (SB) transposon (University of Minnesota, Minneapolis, MN)). The TCR is then expressed in T cells using standard viral transduction or non-viral transfer protocols, and T cells expressing the murine TCR (detected by the murine TCR beta constant chain) are tested against the putative peptide.

[0224] Autologous APCs were transfected with TMG encoding an irrelevant mutation, WT p53 sequence, or mutant p53 sequence containing p53-R248W. Medium alone and PMA and ionomycin were negative and positive controls, respectively. TILs from patient 4273 (fragment cultures 8 and 15) were co-cultured with TMG-transfected APCs overnight at 37°C. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes→live cells (PI negative)→CD3+ (T cells). Results are shown in Figure 56.

[0225] Autologous APCs were pulsed with a 25 amino acid peptide corresponding to the WT or mutant p53-R248W neoepitope for 2 hours at 37°C. TILs (fragment culture 15) from patient 4273 with specificity for p53-R248W were co-cultured with peptide-pulsed APCs overnight at 37°C. DMSO was the peptide vehicle. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 57.

[0226] Autologous APCs were pulsed with a 15 amino acid peptide derived from the p53-R248W neoepitope, which overlaps 14 amino acids. TILs (fragment culture 15) from patient 4273 with specificity for p53-R248W were co-cultured with peptide-pulsed APCs overnight at 37°C. DMSO was the peptide vehicle, and medium alone (T cells only) and PMA and ionomycin were controls. The 25 amino acid peptides (wt p53-R248 and mutant p53-R248W) were further controls for the 15 amino acid peptide. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells) → CD4+CD8-. The results are shown in Figure 58.

[0227] Cos7 cells (2.5×10 4Cells were plated in wells of flat-bottom 96-well plates (cells / well). The following day, cells were co-transfected with individual HLA alleles from patient 4273 and either wild-type or mutant TP53 TMG, with or without the p53-R248W neoantigen, respectively. The following day, TILs with specificity for p53-R248W from patient 4273 (fragment culture 15) were co-cultured overnight at 37° C. with transfected Cos7 cells. IFN-γ secretion was assessed by ELISA. The results are shown in FIG. 59.

[0228] Mock (no TCR) or 4273-TCR1a2 expressing T cells were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of wild-type p53-R248 or mutant p53-R248W sequences. Media alone, PMA and ionomycin were negative and positive controls, respectively. Co-cultures were performed overnight at 37° C. IFN-γ secretion was assessed by ELISPOT. Results are shown in FIG. 60.

[0229] The sequence of TCR4273-TP53-R248W-TCR1a1 isolated from patient 4273 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO: 437), the second underlined region is CDR2 alpha (SEQ ID NO: 438), the third underlined region is CDR3 alpha (SEQ ID NO: 439), the fourth underlined region is CDR1 beta (SEQ ID NO: 440), the fifth underlined region is CDR2 beta (SEQ ID NO: 441), and the sixth underlined region is CDR3 beta (SEQ ID NO: 442). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO: 23), and the second italicized region is the beta chain constant region (SEQ ID NO: 25). The alpha chain variable region (SEQ ID NO: 443) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:444) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:445) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:446) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus. TCR Name: 4273-TP53-R248W-TCR1a1 Recognizing p53 mutation: R248W Methods: Universal screening for p53 "hotspot" mutations

[0230] [ka]

[0231] The sequence of TCR4273-TP53-R248W-TCR1a2 isolated from patient 4273 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO: 447), the second underlined region is CDR2 alpha (SEQ ID NO: 448), the third underlined region is CDR3 alpha (SEQ ID NO: 449), the fourth underlined region is CDR1 beta (SEQ ID NO: 450), the fifth underlined region is CDR2 beta (SEQ ID NO: 451), and the sixth underlined region is CDR3 beta (SEQ ID NO: 452). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO: 23), and the second italicized region is the beta chain constant region (SEQ ID NO: 25). The alpha chain variable region (SEQ ID NO: 453) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:454) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:455) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:456) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus. TCR Name: 4273-TP53-R248W-TCR1a2 Recognizing p53 mutation: R248W Methods: Universal screening for p53 "hotspot" mutations

[0232] [ka]

[0233] Statistics for TCR of patient 4273 are listed in Table 19 below. In Table 19, all 96 wells were sorted with 41BB+ T cells after co-culture with mutant p53 protein (TMG or peptide). 77 wells had productive pairs (i.e., (1) had sequence and (2) did not have a stop codon in the sequence) with a pairing frequency of 80.2%. 43 of these pairs were CDR3A / CDR3B combinations to make 4273-TP53-R248W-TCR1a1 (55.8% of productive pairs). 30 of these pairs were CDR3A / CDR3B combinations to make 4273-TP53-R248W-TCR1a2 (39% of productive pairs). In summary, CDR3A and CDR3B of 4273-TP53-R248W-TCR1a1 were found 50 and 83 times, respectively, out of 96 wells. In summary, CDR3A and CDR3B of 4273-TP53-R248W-TCR1a2 were found 33 and 83 times, respectively, out of 96 wells.

[0234] [Table 19]

[0235] Example 6 This example shows the identification of anti-mutated p53 T cells in patient 4149. This example also shows the isolation and specific reactivity of one anti-mutated p53 TCR from patient 4149.

[0236] For patient 4149, the experiment was carried out as shown in Figures 21-24. The Tran method was used to find the TCR. The TCR was then used to validate the "p53 hotspot mutation universal screening" method.

[0237] TCR (4149-TCRa2b1 or 4149-TCRa2b2) was transferred into autologous PBL from patient 4149 and co-cultured with autologous APCs that were either (1) electroporated with TMG composed of irrelevant, WTp53, or mutant p53 sequences, or (2) pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptide composed of WTp53-Y220 or mutant p53-Y220C sequences. A combination of PMA and ionomycin was the positive control. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 21. 4-1BB expression was assessed by flow cytometry after gating lymphocytes → live cells (PI negative) → CD3+ (T cells) → CD4+mTCR+ (TCR-transferred T cells). Results are shown in Figure 22.

[0238] The percentage of CD4+4-1BB+ cells was also determined by TCRAD and TCRB deep sequencing. The results are shown in Table E.

[0239] [Table E]

[0240] Putative p53 recognized by 4149-F11 Y220C Mapping of minimal epitopes: Autologous DCs were pulsed with peptide (10 μg / mL) and incubated with granulocyte-macrophage colonies. TILs were incubated overnight in stimulatory factors (GM-CSF) and IL-4. TILs were incubated for 2-3 days in 500 CU / mL IL-2. 2×10 4 TIL and 10 5 Target cells were co-cultured overnight at 37°C. IFN-γ was measured by ELISPOT. Results are shown in Table F. 4-1BB expression was measured by FACS gated on lymphocytes \PI(negative)CD3+\CD3+CD4+. Results are shown in Figure 23.

[0241] [Table F]

[0242] Cos7 cells (2.5×10 4 Cells (10 cells / well) were plated into wells of a flat-bottom 96-well plate. After 20 hours, cells were co-transfected with individual HLA alleles with or without TMG. After 20 hours, autologous DC cells were transfected with TMG in parallel. All HLA class II alleles were co-transfected with or without TMG into one set of wells. Cells not transfected with TMG were pulsed with 10 μg / mL p53-Y220C 15-mer peptide for 2–3 hours at 37°C. After washing, 4149-TCRa2b2-transfected T cells (10 5 ) were added to the wells and co-cultured overnight at 37° C. IFN-γ secretion was measured by ELISA. The results are shown in FIG. 24. Predictions by NetMHCIIpan: cbs.dtu.dk / services / NetMHCIIpan / (Table G).

[0243] [Table G]

[0244] In the NCI HLA database, 1367 (37%) of 3719 patients with DRB type and in the NCI-SB, 5 (56%) of 9 patients with endometrial and ovarian cancer had DRB3. * 02 expression was detected. * The frequency of the 02 allele is very high, as described in Example 1.

[0245] The sequence of TCR4149TCRa2b2 isolated from patient 4149 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:57), the second underlined region is CDR2 alpha (SEQ ID NO:58), and the third underlined region is CDR3 alpha (SEQ ID NO:59). R3 alpha (SEQ ID NO:59), the fourth underlined region is CDR1 beta (SEQ ID NO:60), the fifth underlined region is CDR2 beta (SEQ ID NO:61), and the sixth underlined region is CDR3 beta (SEQ ID NO:62). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:63) contains the sequence that begins at the amino terminus and ends just before the start of the alpha chain constant region. The beta chain variable region (SEQ ID NO:64) contains the sequence that begins just after the linker and ends just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:65) contains the sequence that begins at the amino terminus and ends just before the start of the linker. The full-length beta chain (SEQ ID NO:66) contains the sequence that begins just after the linker and ends at the carboxyl terminus.

[0246] Cancer-reactive T cells were identified using the screening method described below: p53-reactive cells in this patient were identified by the Tran screening method described in US Patent Application Publication No. 2017 / 0224800. TCR name: 4149TCRa2b2 Recognizing p53 mutation: Y220C Screening method: Used to validate p53 "hot spot" mutation universal screening Co-culture to identify TCR: Co-culture 4149-F11 TIL fragments with p53-Y220C long peptide and sort CD4+41BB+ T cells Methods for identifying TCRs: Pairing frequency. CD4+41BB+ sorted T cells were expanded by REP (Rapid Expansion Protocol) and the resulting T cell cultures were subjected to TCRAD (alpha) and TCRB (beta) deep sequencing by Adaptive Biotechnologies. The top two TCR alphas were paired with the top two TCR betas in a matrix of four total TCRs. The second TCR alpha and the second TCR beta were reactive TCRs and were therefore named TCRa2b2. Abundance of TCR among all TCRs: TCR orientation: alpha-beta Expression vector: SB transposon

[0247] [ka]

[0248] Statistics for TCR4149TCRa2b2 for patient 4149 are listed in Table 20 below.

[0249] [Table 20]

[0250] Example 7 This example shows the identification of anti-mutated p53 T cells in patient 4213 by co-culturing autologous APCs induced to express mutant p53 in autologous T cells ("p53 hotspot mutation universal screening"). This example also shows the isolation of 12 anti-mutated p53 TCRs from patient 4213.

[0251] For patient 4213, the experiment was performed as depicted in Figures 25-26.

[0252] TIL fragments (F2 and F24) from patient 4213 were co-cultured with autologous APC pulsed with peptide vehicle (DMSO) or a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248Q sequence. Co-cultures were performed overnight at 37°C. Expression of 4-1BB was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 25. CD8+4-1BB+ T cells were sorted into wells of a 96-well plate. TCRs were identified using single cell RT-PCR.

[0253] CD4+ T cells were derived from peripheral blood lymphocytes of patient 4213. CD4+ T cell cultures were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or a purified (>95% by HPLC) 25 amino acid peptide composed of the mutant p53-R248Q sequence. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 26. CD4+41BB+ T cells were sorted into wells of a 96-well plate. TCRs were identified using single-cell RT-PCR.

[0254] The sequence of 4213-F2-TCR1 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:317), the second underlined region is CDR2 alpha (SEQ ID NO:318), the third underlined region is CDR3 alpha (SEQ ID NO:319), the fourth underlined region is CDR1 beta (SEQ ID NO:320), the fifth underlined region is CDR2 beta (SEQ ID NO:321), and the sixth underlined region is CDR3 beta (SEQ ID NO:322). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:323) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:324) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO:325) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO:326) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0255] Cancer-reactive T cells were identified using the screening method described below. The method used to isolate is described below. TCR name: 4213-F2-TCR1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4213-F2 with R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 8.3% (observed 2 times out of 24 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0256] [ka]

[0257] Statistics for TCR4213-F2-TCR1 for patient 4213 are listed in Table 21 below.

[0258] [Table 21]

[0259] The sequence of 4213-F2-TCR2 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:327), the second underlined region is CDR2 alpha (SEQ ID NO:328), the third underlined region is CDR3 alpha (SEQ ID NO:329), the fourth underlined region is CDR1 beta (SEQ ID NO:330), the fifth underlined region is CDR2 beta (SEQ ID NO:331), and the sixth underlined region is CDR3 beta (SEQ ID NO:332). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:333) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:334) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:335) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:336) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0260] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-F2-TCR2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4213-F2 with R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 12.5% ​​(observed 3 times out of 24 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0261] [ka]

[0262] Statistics for TCR4213-F2-TCR2 for patient 4213 are listed in Table 22 below.

[0263] [Table 22]

[0264] The sequence of 4213-F2-TCR3 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:337), the second underlined region is CDR2 alpha (SEQ ID NO:338), the third underlined region is CDR3 alpha (SEQ ID NO:339), the fourth underlined region is CDR1 beta (SEQ ID NO:340), the fifth underlined region is CDR2 beta (SEQ ID NO:341), and the sixth underlined region is CDR3 beta (SEQ ID NO:342). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:343) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:344) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO:345) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO:346) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0265] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-F2-TCR3 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4213-F2 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 70.8% (observed 17 times out of 24 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0266] [ka]

[0267] Statistics for TCR4213-F2-TCR3 for patient 4213 are listed in Table 23 below.

[0268] [Table 23]

[0269] The sequence of 4213-F24-TCRa1 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:347), the second underlined region is CDR2 alpha (SEQ ID NO:348), the third underlined region is CDR3 alpha (SEQ ID NO:349), the fourth underlined region is CDR1 beta (SEQ ID NO:350), the fifth underlined region is CDR2 beta (SEQ ID NO:351), and the sixth underlined region is CDR3 beta (SEQ ID NO:352). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:353) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:354) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:355) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:356) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0270] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-F24-TCRa1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4213-F24 with R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 15.9% (7 observations out of 44 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0271] [ka]

[0272] Statistics for TCR4213-F24-TCRa1 for patient 4213 are listed in Table 24 below.

[0273] [Table 24]

[0274] The sequence of 4213-F24-TCRa2 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:357), the second underlined region is CDR2 alpha (SEQ ID NO:358), the third underlined region is CDR3 alpha (SEQ ID NO:359), the fourth underlined region is CDR1 beta (SEQ ID NO:360), the fifth underlined region is CDR2 beta (SEQ ID NO:361), and the sixth underlined region is CDR3 beta (SEQ ID NO:362). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:363) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:364) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:365) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:366) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0275] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-F24-TCRa2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4213-F24 with R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 84.1% (observed 37 times out of 44 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0276] [ka]

[0277] Statistics for TCR4213-F24-TCRa2 for patient 4213 are listed in Table 25 below.

[0278] [Table 25]

[0279] The sequence of 4213-PBL-TCR1 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:367), the second underlined region is CDR2 alpha (SEQ ID NO:368), the third underlined region is CDR3 alpha (SEQ ID NO:369), the fourth underlined region is CDR1 beta (SEQ ID NO:370), the fifth underlined region is CDR2 beta (SEQ ID NO:371), and the sixth underlined region is CDR3 beta (SEQ ID NO:372). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:373) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:374) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:375) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:376) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0280] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR:C after in vitro sensitization with R248Q long peptide D4+ memory T cells were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 9.5% (observed 6 times out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon (University of Minnesota, Minneapolis, MN)

[0281] [ka]

[0282] Statistics for TCR4213-PBL-TCR1 for patient 4213 are listed in Table 26 below.

[0283] [Table 26]

[0284] The sequence of 4213-PBL-TCR2 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:377), the second underlined region is CDR2 alpha (SEQ ID NO:378), the third underlined region is CDR3 alpha (SEQ ID NO:379), the fourth underlined region is CDR1 beta (SEQ ID NO:380), the fifth underlined region is CDR2 beta (SEQ ID NO:381), and the sixth underlined region is CDR3 beta (SEQ ID NO:382). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:383) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:384) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO:385) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO:386) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0285] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: CD4+ memory T cells after in vitro sensitization with R248Q long peptide were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 7.9% (5 observations out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0286] [ka]

[0287] Statistics for TCR4213-PBL-TCR2 for patient 4213 are listed in Table 27 below.

[0288] [Table 27]

[0289] The sequence of 4213-PBL-TCR3 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:387), the second underlined region is CDR2 alpha (SEQ ID NO:388), the third underlined region is CDR3 alpha (SEQ ID NO:389), the fourth underlined region is CDR1 beta (SEQ ID NO:390), the fifth underlined region is CDR2 beta (SEQ ID NO:391), and the sixth underlined region is CDR3 beta (SEQ ID NO:392). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:393) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:394) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:395) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:396) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0290] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR3 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: CD4+ memory T cells after in vitro sensitization with R248Q long peptide were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 6.3% (4 observations out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0291] [ka]

[0292] Statistics for TCR4213-PBL-TCR3 for patient 4213 are listed in Table 28 below.

[0293] [Table 28]

[0294] The sequence of 4213-PBL-TCR4a1 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:397), the second underlined region is CDR2 alpha (SEQ ID NO:398), the third underlined region is CDR3 alpha (SEQ ID NO:399), the fourth underlined region is CDR1 beta (SEQ ID NO:400), the fifth underlined region is CDR2 beta (SEQ ID NO:401), and the sixth underlined region is CDR3 beta (SEQ ID NO:402). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:403) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:404) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO:405) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO:406) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0295] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR4a1 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: CD4+ memory T cells after in vitro sensitization with R248Q long peptide were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 3.2% (observed 2 times out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0296] [ka]

[0297] Statistics for TCR4213-PBL-TCR4a1 for patient 4213 are listed in Table 29 below.

[0298] [Table 29]

[0299] The sequence of 4213-PBL-TCR4a2 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:407), the second underlined region is CDR2 alpha (SEQ ID NO:408), the third underlined region is CDR3 alpha (SEQ ID NO:409), the fourth underlined region is CDR1 beta (SEQ ID NO:410), the fifth underlined region is CDR2 beta (SEQ ID NO:411), and the sixth underlined region is CDR3 beta (SEQ ID NO:412). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:413) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:414) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:415) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:416) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0300] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR4a2 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: CD4+ memory T cells after in vitro sensitization with R248Q long peptide were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 3.2% (observed 2 times out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0301] [ka]

[0302] Statistics for TCR4213-PBL-TCR4a2 for patient 4213 are set forth in Table 30 below.

[0303] [Table 30]

[0304] The sequence of 4213-PBL-TCR4a3 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:417), the second underlined region is CDR2 alpha (SEQ ID NO:418), the third underlined region is CDR3 alpha (SEQ ID NO:419), the fourth underlined region is CDR1 beta (SEQ ID NO:420), the fifth underlined region is CDR2 beta (SEQ ID NO:421), and the sixth underlined region is CDR3 beta (SEQ ID NO:422). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:423) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:424) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:425) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:426) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0305] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR4a3 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: CD4+ memory T cells after in vitro sensitization with R248Q long peptide were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 4.8% (observed 3 times out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0306] [ka]

[0307] Statistics for TCR4213-PBL-TCR4a3 for patient 4213 are listed in Table 31 below.

[0308] [Table 31]

[0309] The sequence of 4213-PBL-TCR4a4 isolated from patient 4213 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO: 427), the second underlined region is CDR2 alpha (SEQ ID NO: 428), the third underlined region is CDR3 alpha (SEQ ID NO: 429), the fourth underlined region is CDR1 beta (SEQ ID NO: 430), the fifth underlined region is CDR2 beta (SEQ ID NO: 431), and the sixth underlined region is CDR3 beta (SEQ ID NO: 432). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO: 23), and the second italicized region is the beta chain constant region (SEQ ID NO: 25). The alpha chain variable region (SEQ ID NO: 433) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:434) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:435) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:436) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0310] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4213-PBL-TCR4a4 Recognizing p53 mutation: R248Q Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: CD4+ memory T cells after in vitro sensitization with R248Q long peptide were co-cultured with R248Q long peptide, and CD4+41BB+ T cells were sorted. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 3.2% (observed 2 times out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0311] [ka]

[0312] Statistics for TCR4213-PBL-TCR4a4 for patient 4213 are listed in Table 32 below.

[0313] [Table 32]

[0314] Example 8 This example shows the identification of anti-mutant p53 T cells in patient 4268 by co-culturing autologous APCs induced to express mutant p53 in autologous T cells ("p53 hotspot mutation universal screening"). This example also shows the isolation of five anti-mutant p53 TCRs from patient 4268.

[0315] For patient 4268, the study was performed as described in Figures 27-30.

[0316] TIL fragments (F1-F24, n=24) from patient 4268 were co-cultured with autologous APCs electroporated with irrelevant, WT p53, or TMG composed of mutant p53 sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 27.

[0317] TIL fragments (F1-F24, n=24) from patient 4268 were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 or mutant p53-R248Q sequences. Co-cultures were performed overnight at 37° C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in FIG. 28.

[0318] TIL fragments (F1-F24, n=24) derived from patient 4268 were cultured in a 30-well plate with peptide vehicle (D MSO) or autologous APC pulsed with a purified (>95% by HPLC) 25 amino acid peptide composed of the wt p53-R248 or mutant p53-R248Q sequence. Co-culture was performed overnight at 37°C. Expression of 4-1BB was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 29. The source of TCR after sorting CD4+41BB+ T cells was the TIL fragments F18 and F19.

[0319] TIL fragments (F1-F24, n=24) from patient 4268 were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 or mutant p53-R248Q sequences. Co-cultures were performed overnight at 37°C. Expression of 4-1BB was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 30. The source of TCR after sorting CD8+4-1BB+ T cells was TIL fragments F7, F8, and F15.

[0320] The sequence of 4268-TCR1 isolated from patient 4268 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:137), the second underlined region is CDR2 alpha (SEQ ID NO:138), the third underlined region is CDR3 alpha (SEQ ID NO:139), the fourth underlined region is CDR1 beta (SEQ ID NO:140), the fifth underlined region is CDR2 beta (SEQ ID NO:141), and the sixth underlined region is CDR3 beta (SEQ ID NO:142). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:143) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 144) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 145) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO: 146) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0321] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4268-TCR1 Recognizing p53 mutation: R248Q Methods: Universal screening for p53 "hotspot" mutations Co-culture to identify TCR: Co-culture 4268-F7 and 4268-F8 with p53-R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 78.7% (48 observations out of 61 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0322] [ka]

[0323] Statistics for TCR4268-TCR1 for patient 4268 are listed in Table 33 below.

[0324] [Table 33]

[0325] The sequence of 4268-TCR2 isolated from patient 4268 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:147), the second underlined region is CDR2 alpha (SEQ ID NO:148), the third underlined region is CDR3 alpha (SEQ ID NO:149), the fourth underlined region is CDR1 beta (SEQ ID NO:150), the fifth underlined region is CDR2 beta (SEQ ID NO:151), and the sixth underlined region is CDR3 beta (SEQ ID NO:152). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:153) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 154) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 155) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO: 156) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0326] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4268-TCR2 Recognizing p53 mutation: R248Q Methods: Universal screening for p53 "hotspot" mutations Co-culture to identify TCR: Co-culture 4268-F7 and 4268-F8 with p53-R248Q long peptide and sort CD8+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 6.6% (4 observations out of 61 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0327] [ka]

[0328] Statistics for TCR4268-TCR2 for patient 4268 are listed in Table 34 below.

[0329] [Table 34]

[0330] The sequence of 4268-TCR3 isolated from patient 4268 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:157), the second underlined region is CDR2 alpha (SEQ ID NO:158), the third underlined region is CDR3 alpha (SEQ ID NO:159), the fourth underlined region is CDR1 beta (SEQ ID NO:160), the fifth underlined region is CDR2 beta (SEQ ID NO:161), and the sixth underlined region is CDR3 beta (SEQ ID NO:162). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:163) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 164) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 165) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO: 166) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0331] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4268-TCR3 Recognizing p53 mutation: R248Q Methods: Universal screening for p53 "hotspot" mutations Co-culture to identify TCR: Co-culture 4268-F15 with p53-R248Q long peptide and sort CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 88.6% (observed 31 out of 35 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0332] [ka]

[0333] Statistics for TCR4268-TCR3 for patient 4268 are listed in Table 35 below.

[0334] [Table 35]

[0335] The sequence of 4268-TCR4 isolated from patient 4268 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:167), the second underlined region is CDR2 alpha (SEQ ID NO:168), the third underlined region is CDR3 alpha (SEQ ID NO:169), the fourth underlined region is CDR1 beta (SEQ ID NO:170), the fifth underlined region is CDR2 beta (SEQ ID NO:171), and the sixth underlined region is CDR3 beta (SEQ ID NO:172). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:173) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 174) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 175) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO: 176) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0336] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4268-TCR4 Recognizing p53 mutation: R248Q Methods: Universal screening for p53 "hotspot" mutations Co-culture to identify TCR: Co-culture 4268-F18 with p53-R248Q long peptide and sort CD4+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 95.2% (40 observations out of 42 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0337] [ka]

[0338] Statistics for TCR4268-TCR4 for patient 4268 are listed in Table 36 below.

[0339] [Table 36]

[0340] The sequence of 4268-TCR5 isolated from patient 4268 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:177), the second underlined region is CDR2 alpha (SEQ ID NO:178), the third underlined region is CDR3 alpha (SEQ ID NO:179), the fourth underlined region is CDR1 beta (SEQ ID NO:180), the fifth underlined region is CDR2 beta (SEQ ID NO:181), and the sixth underlined region is CDR3 beta (SEQ ID NO:182). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:183) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 184) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 185) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO: 186) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0341] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4268-TCR5 Recognizing p53 mutation: R248Q Methods: Universal screening for p53 "hotspot" mutations Co-culture to identify TCR: Co-culture 4268-F19 with p53-mut-TMG and sort CD4+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 11.8% (2 observations out of 17 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0342] [ka]

[0343] Statistics for TCR4268-TCR5 for patient 4268 are listed in Table 37 below.

[0344] [Table 37]

[0345] Example 9 This example shows the identification of anti-mutant p53 T cells in patient 4266 by co-culturing autologous APCs induced to express mutant p53 in autologous T cells ("p53 hotspot mutation universal screen"). This example also shows the isolation of four anti-mutant p53 TCRs from patient 4266.

[0346] For patient 4266, the study was performed as described in Figures 31-34 and 53-55.

[0347] TIL fragments (F1-F24, n=24) from patient 4266 were co-cultured with autologous APCs electroporated with TMG composed of irrelevant, WT p53, or mutant p53 sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 31. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 32.

[0348] TIL fragments (F1-F24, n=24) from patient 4266 were co-cultured with autologous APC pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 or mutant p53-R248W sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 33. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 34.

[0349] Cos7 cells (2.5×10 4 Cells were plated with 1000 ng / well of 1000 ng / ml ... TIL culture from subject 4266 (10 5 ) were added to the wells and co-cultured overnight at 37°C. After gating on lymphocytes → live cells (PI negative) → CD3+ (T cells) → CD4-CD8+, 4-1BB expression was evaluated by flow cytometry. The results are shown in Figure 53.

[0350] T cells expressing mock (no TCR), 4266-TCR1, 4266-TCR2, 4266-TCR3, or 4266-TCR4 with putative specificity for p53-R248W identified from 4266-TILs were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R248 or mutant p53-R248W sequences. Media alone and PMA and ionomycin were negative and positive controls, respectively. Co-cultures were performed overnight at 37°C. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes→live cells (PI negative)→CD3+ (T cells)→CD4−CD8+. Results are shown in Figure 54.

[0351] Tumor cell (TC) lines were established from xenograft tumor fragments excised from patient 4266 and then serially passaged in immunocompromised mice (TC#4266). T cells (10 5 TC#4266 cells were co-cultured with TC#4266 cells at 37°C overnight. TC#4266 cells were incubated with W6 / 32 pan-HLA class I specific blocking antibody, IVA12 pan-HLA class II specific blocking antibody, or mutant p53-R248W peptide SSCMGGMNWR (SEQ ID NO:591) for 2 hours at 37°C. Antibodies were maintained in the co-cultures at a final concentration of 5 μg / mL. Peptides were incubated at 1 μg / mL and excess peptide was washed after incubation. Medium alone (no TC) and PMA and ionomycin were negative and positive controls, respectively. 4-1BB expression was evaluated by flow cytometry after gating lymphocytes→live cells (PI negative)→CD3+(T cells)→CD4-CD8+. The results are shown in Figure 55.

[0352] The sequence of 4266-TCR1 isolated from patient 4266 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:97), the second underlined region is CDR2 alpha (SEQ ID NO:98), the third underlined region is CDR3 alpha (SEQ ID NO:99), the fourth underlined region is CDR1 beta (SEQ ID NO:100), the fifth underlined region is CDR2 beta (SEQ ID NO:101), and the sixth underlined region is CDR3 beta (SEQ ID NO:102). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:103) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 104) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 105) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO: 106) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0353] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4266-TCR1 Recognizing p53 mutation: R248W Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: 4266-F1, 4266-F3, 4266-F5, and and co-culture of 4266-F6 with p53mutTMG or R248W long peptide (both co-cultures detected the same TCR), and sorting of CD8+41BB+ T cells. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 17.0% (observed 9 times out of 53 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0354] [ka]

[0355] Statistics for TCR4266-TCR1 for patient 4266 are listed in Table 38 below.

[0356] [Table 38]

[0357] The sequence of 4266-TCR2 isolated from patient 4266 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:107), the second underlined region is CDR2 alpha (SEQ ID NO:108), the third underlined region is CDR3 alpha (SEQ ID NO:109), the fourth underlined region is CDR1 beta (SEQ ID NO:110), the fifth underlined region is CDR2 beta (SEQ ID NO:111), and the sixth underlined region is CDR3 beta (SEQ ID NO:112). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:113) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:114) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:115) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:116) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0358] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4266-TCR2 Recognizing p53 mutation: R248W Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: 4266-F1, 4266-F3, 4266-F5, and and co-culture of 4266-F6 with p53mutTMG or R248W long peptide (both co-cultures detected the same TCR), and sorting of CD8+41BB+ T cells. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 24.5% (observed 13 times out of 53 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0359] [ka]

[0360] Statistics for TCR4266-TCR2 for patient 4266 are listed in Table 39 below.

[0361] [Table 39]

[0362] The sequence of 4266-TCR3 isolated from patient 4266 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:117), the second underlined region is CDR2 alpha (SEQ ID NO:118), the third underlined region is CDR3 alpha (SEQ ID NO:119), the fourth underlined region is CDR1 beta (SEQ ID NO:120), the fifth underlined region is CDR2 beta (SEQ ID NO:121), and the sixth underlined region is CDR3 beta (SEQ ID NO:122). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:123) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:124) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:125) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:126) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0363] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4266-TCR3 Recognizing p53 mutation: R248W Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: 4266-F1, 4266-F3, 4266-F5, and and co-culture of 4266-F6 with p53mutTMG or R248W long peptide (both co-cultures detected the same TCR), and sorting of CD8+41BB+ T cells. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 34.0% (observed 18 times out of 53 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0364] [ka]

[0365] Statistics for TCR4266-TCR3 for patient 4266 are listed in Table 40 below.

[0366] [Table 40]

[0367] The sequence of 4266-TCR4 isolated from patient 4266 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:127), the second underlined region is CDR2 alpha (SEQ ID NO:128), the third underlined region is CDR3 alpha (SEQ ID NO:129), the fourth underlined region is CDR1 beta (SEQ ID NO:130), the fifth underlined region is CDR2 beta (SEQ ID NO:131), and the sixth underlined region is CDR3 beta (SEQ ID NO:132). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:23), and the second italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:133) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 134) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 135) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO: 136) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0368] Cancer-reactive T cells were identified using the screening methods described below. The methods used to isolate TCRs are described below. TCR name: 4266-TCR4 Recognizing p53 mutation: R248W Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4266-F1, 4266-F3, 4266-F5, and 4266-F6 with p53mutTMG or R248W long peptide (both co-cultures detected the same TCR), and sort CD8+41BB+ T cells. Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 9.4% (5 observations out of 53 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0369] [ka]

[0370] Statistics for TCR4266-TCR4 for patient 4266 are listed in Table 41 below.

[0371] [Table 41]

[0372] Example 10 This example outlines the response of T cells to p53 "hot spot" mutations.

[0373] A summary of the response to p53 "hot spot" mutations by T cells is provided in Table 42. Numbers 1-15 in Table 42 were retrospective studies. Numbers 16-33 in Table 42 were prospective studies.

[0374] [Table 42]

[0375] Example 11 This example demonstrates treatment of a patient with p53 mutation-responsive TILs.

[0376] A summary of the treatment of patients with p53 mutation-responsive TILs is provided in Table 43.

[0377] [Table 43]

[0378] Example 12 This example shows the isolation and specific reactivity of a TCR from patient 4141.

[0379] 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 co-cultured with TMG-transfected APCs overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes→live cells (PI negative)→CD3+ (T cells)→CD4-CD8+. Results are shown in Figure 38.

[0380] Cos7 cells (2.5×10 4 The following day, individual HLA alleles from patient 4141 and either no additional genes, WT TP53 TMG, or mutant TP53 containing the p53-R175H sequence were plated into wells of a flat-bottom 96-well plate. The cells were co-transfected with either TMG or TIL. The following day, TILs with specificity for p53-R175H from patient 4141 (fragment culture 12) were co-cultured with the transfected Cos7 cells and incubated overnight at 37° C. IFN-γ secretion was assessed by ELISPOT. The results are shown in FIG. 39.

[0381] Mock (no TCR) or 4141-TCR1a2-expressing T cells were transfected with T2 tumor cells (HLA-A *T2 cells were co-cultured with WT p53-R175H peptide HMTEVVRRC (SEQ ID NO: 532) or mutant p53-R175H peptide HMTEVVRHC (SEQ ID NO: 530) for 2 h at 37° C. Peptide vehicle (DMSO) or purified (>95% by HPLC) peptides consisting of WT p53-R175H peptide HMTEVVRHC (SEQ ID NO: 530) were pulsed for 2 h at 37° C. Media alone and PMA and ionomycin were negative and positive controls, respectively. Co-culture was performed overnight at 37° C. IFN-γ secretion was assessed by ELISA. Results are shown in FIG. 40.

[0382] T cells expressing 4141-TCR1a2 were incubated overnight at 37°C with Saos2 cells (p53-NULL and HLA-A * 02:01+), which were either unengineered or made to overexpress full-length p53-R175H protein. To capture cytokines in T cells, inhibitors of secretion (monensin and brefeldin A) were added to the co-cultures. After 6 hours of co-culture, cells were fixed, permeabilized, and then stained for IL-2, CD107a, IFN-γ, and tumor necrosis factor alpha (TNFα). Flow cytometry was used to analyze the co-cultures based on the lymphocyte gate. The results are shown in Figure 41.

[0383] The sequence of TCR4141-TCR1a2 isolated from patient 4141 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO: 467), the second underlined region is CDR2 alpha (SEQ ID NO: 468), the third underlined region is CDR3 alpha (SEQ ID NO: 469), the fourth underlined region is CDR1 beta (SEQ ID NO: 470), the fifth underlined region is CDR2 beta (SEQ ID NO: 471), and the sixth underlined region is CDR3 beta (SEQ ID NO: 472). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO: 23), and the second italicized region is the beta chain constant region (SEQ ID NO: 25). The alpha chain variable region (SEQ ID NO: 473) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:474) contains the sequence beginning immediately after the linker and ending just before the beginning of the beta chain constant region. The full-length alpha chain (SEQ ID NO:475) contains the sequence beginning at the amino terminus and ending just before the beginning of the linker. The full-length beta chain (SEQ ID NO:476) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0384] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR name: 4141-TCR1a2 Recognizing p53 mutation: R175H Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4141 infusion bag TIL with p53mutTMG and sort CD8+41BB+ T cells Method for identifying TCR: Single cell RT-PCR followed by TA for the alpha chain TOPO cloning kit (Thermo Fisher Scientific, Waltham, MA) TCR orientation: alpha-beta Expression vector: SB transposon

[0385] [ka]

[0386] Statistics for 4141-TCR1a2 from patient 4141 are set forth in Table 44 below.

[0387] [Table 44]

[0388] Example 13 This example shows the isolation and specific reactivity of a TCR isolated from patient 4259.

[0389] TIL fragment cultures (#6) from patient 4259 were co-cultured with autologous APCs (1) electroporated with TMG composed of irrelevant, WTp53, or mutant p53 sequences, or (2) pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WTp53-Y220 or mutant p53-Y220C sequences. Co-cultures were performed overnight at 37°C. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 42.

[0390] Autologous APCs were pulsed with decreasing concentrations of a 25 amino acid peptide corresponding to the sequence of WTp53-Y220 or mutant p53-Y220C for 2 hours at 37°C. A TIL fragment culture (#6) from patient 4259 was co-cultured with peptide-pulsed APCs. After gating on lymphocytes→live cells (PI negative)→CD3+ (T cells), 4-1BB expression was assayed by flow cytometry. The results are shown in Figure 43.

[0391] Autologous antigen-presenting cells were pulsed with DMSO, WT p53-Y220 peptide RNTFRHSVVVPYE (SEQ ID NO: 533), or mutant p53-Y220C peptide RNTFRHSVVVPCE (SEQ ID NO: 534) for 2 hours at 37°C. Excess peptide was washed away. TILs from patient 4259 with specificity for p53-Y220C (fragment culture 6) were co-cultured with peptide-pulsed APCs overnight at 37°C. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 44.

[0392] Cos7 cells (2.5×10 4 Cells were plated in wells of a flat-bottom 96-well plate (100 μg / well). The following day, individual HLA alleles from patient 4259 were co-transfected into the cells. The following day, DMSO or p53-Y220C peptide RNTFRHSVVVPCE (SEQ ID NO: 534) was pulsed into transfected Cos7 cells for 2 hours. Excess peptide was washed away. TIL fragment culture #6 from patient 4259 was added (100 μg / well). 0 5 Co-cultures were incubated overnight at 37° C. Expression of 4-1BB was assayed by flow cytometry after gating on lymphocytes→viable cells→CD3+ (T cells)→CD8-CD4+. The results are shown in FIG.

[0393] A 25 amino acid peptide corresponding to the sequence of WTp53-Y220 or mutant p53-Y220C was pulsed onto autologous APCs for patient 4259 at 37°C for 2 hours. Excess peptide was washed away. T cells expressing 4259-F6-TCR were co-cultured with peptide-pulsed APCs overnight at 37°C. After gating on lymphocytes → live cells (PI negative) → CD3+ (T cells), 4-1BB expression was evaluated by flow cytometry. Transduced TCR was measured by mouse TCR beta (mTCR). The results are shown in Table 45.

[0394] [Table 45]

[0395] A tumor cell (TC) line was established from a xenograft tumor fragment excised from patient 4259 and then serially passaged in immunocompromised mice (TC#4259). TC#4259 was cultured with mock (no TCR) or T cells expressing the p53-Y220C-specific TCR (4259-F6-TCR) (10 5 TC#4259 cells were co-cultured with W6 / 32 pan-HLA class I specific blocking antibody, IVA12 pan-HLA class II specific blocking antibody, or mutant p53-Y220C peptide RNTFRHSVVVPCE (SEQ ID NO: 534) at 37°C overnight. TC#4259 cells were incubated with nothing, W6 / 32 pan-HLA class I specific blocking antibody, IVA12 pan-HLA class II specific blocking antibody, or mutant p53-Y220C peptide RNTFRHSVVVPCE (SEQ ID NO: 534) for 2 hours at 37°C. Antibodies were maintained in the co-cultures at a final concentration of 5 μg / mL. Peptides were incubated at 10 μg / mL and excess peptide was washed after incubation. Medium alone (no TC) and PMA and ionomycin were negative and positive controls, respectively. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 46.

[0396] The sequence of TCR4259-F6-TCR isolated from patient 4259 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO: 477), the second underlined region is CDR2 alpha (SEQ ID NO: 478), the third underlined region is CDR3 alpha (SEQ ID NO: 479), the fourth underlined region is CDR1 beta (SEQ ID NO: 480), the fifth underlined region is CDR2 beta (SEQ ID NO: 481), and the sixth underlined region is CDR3 beta (SEQ ID NO: 482). The bolded region is the linker (SEQ ID NO: 26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO: 23), and the second italicized region is the beta chain constant region (SEQ ID NO: 25). The alpha chain variable region (SEQ ID NO: 483) includes the sequence beginning at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:484) contains the sequence beginning immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:485) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:486) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[0397] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR name: 4259-F6-TCR Recognition of p53 mutation: Y220C Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4259-F6 with p53-Y220C peptide and sort CD4+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 81.1% (observed 36 times out of 44 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0398] [ka]

[0399] Statistics for 4259-F6-TCR from patient 4259 are listed in Table 46 below.

[0400] [Table 46]

[0401] Example 14 This example shows the isolation and specific reactivity of a TCR from patient 4285.

[0402] TIL fragments (F1-F22 and F24, n=23) from patient 4285 were co-cultured with autologous APCs electroporated with TMG composed of irrelevant, WT p53, or mutant p53 sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 47. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 49.

[0403] TIL fragments (F1-F22 and F24, n=23) from patient 4285 were co-cultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25 amino acid peptides composed of WT p53-R175 or mutant p53-R175H sequences. Co-cultures were performed overnight at 37°C. IFN-γ secretion was evaluated using an ELISPOT assay. Results are shown in Figure 48. 4-1BB expression was evaluated by flow cytometry after gating lymphocytes → live cells (PI negative) → CD3+ (T cells). Results are shown in Figure 50. The source of 4285-TCR1 after sorting CD4+41BB+ T cells was the TIL fragment F6.

[0404] Autologous APCs were pulsed with a 15-amino acid peptide derived from the p53-R175H sequence overlapping 14 amino acids (underlined amino acid substitutions in Table 45). TILs from patient 4285 with specificity for p53-R175H (fragment cultures 10, 6, and 9) were co-cultured overnight at 37° C. with peptide-pulsed APCs. DMSO was the peptide vehicle. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Table 47.

[0405] [Table 47]

[0406] Cos7 cells (2.5×10 4 1000 cells / well) were plated into wells of a flat-bottom 96-well plate. The following day, cells were co-transfected with individual HLA alleles from patient 4285. The following day, cells were pulsed with DMSO or mutant p53-R175H peptide YKQSQHMTEVVRHCPHHERCSDSDG (SEQ ID NO: 2) at 10 μg / mL for 2 hours at 37° C. Selected TIL fragment cultures with specificity for p53-R175H from patient 4285 (4285-F6, 4285-F9, and 4285-F10) were co-cultured overnight at 37° C. with transfected Cos7 cells. 4-1BB expression was assayed by flow cytometry after gating on lymphocytes→live cells→CD3+ (T cells)→CD8-CD4+. The results are shown in FIG. 51.

[0407] Autologous APCs were pulsed with decreasing concentrations of 25 or 15 amino acid peptides corresponding to the sequences of WT or mutant p53-R175H for 2 hours at 37° C. T cells transferred with 4285-TCR1 from patient 4285 were co-cultured with peptide-pulsed APCs overnight at 37° C. Expression of 4-1BB was assayed by flow cytometry after gating on lymphocytes→live cells→CD3+(T cells)→CD8-CD4+. Results are shown in FIG. 52.

[0408] The sequence of 4285-TCR1 isolated from patient 4285 is set forth below. Starting from the amino terminus, the first underlined region is CDR1 alpha (SEQ ID NO:487), the second underlined region is CDR2 alpha (SEQ ID NO:488), the third underlined region is CDR3 alpha (SEQ ID NO:489), the fourth underlined region is CDR1 beta (SEQ ID NO:490), the fifth underlined region is CDR2 beta (SEQ ID NO:491), and the sixth underlined region is CDR3 beta (SEQ ID NO:492). The bolded region is the linker (SEQ ID NO:26). Starting from the amino terminus, the first italicized region is the alpha chain constant region (SEQ ID NO:493). The first italicized region is the beta chain constant region (SEQ ID NO:25). The alpha chain variable region (SEQ ID NO:493) contains sequence starting at the amino terminus and ending just before the beginning of the alpha chain constant region. The beta chain variable region (SEQ ID NO:494) contains sequence starting just after the linker and ending just before the beginning of the beta chain constant region. The full length alpha chain (SEQ ID NO:495) contains sequence starting at the amino terminus and ending just before the beginning of the linker. The full length beta chain (SEQ ID NO:496) contains sequence starting just after the linker and ending at the carboxyl terminus.

[0409] Cancer-reactive T cells were identified as follows. TCRs were isolated as follows. TCR name: 4285-TCR1 Recognizing p53 mutation: R175H Screening method: Universal screening for p53 "hot spot" mutations Co-culture to identify TCR: Co-culture 4285-F6 with p53-R175H peptide and sort CD4+41BB+ T cells Method for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 81.8% (observed 36 times out of 44 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0410] [ka]

[0411] Statistics for 4285-TCR1 from patient 4185 are listed in Table 48.

[0412] [Table 48]

[0413] Example 15 This example shows the specific reactivity of three anti-mutated p53 TCRs from patient 4196 in Example 2.

[0414] HLA-A with various target cell lines * The expression of 0201 and p53 R175H is presented in Table 49.

[0415] [Table 49]

[0416] The target cells in Table 49 were co-cultured with cells transduced with one of the TCRs in Example 2. Mock-transduced cells (no TCR) were used as control effector cells. IFN-γ secretion (pg / mL) (Table 50) and 4-1BB expression (% of 4-1BB (mTCRβ+)) (Table 51) were measured.

[0417] [Table 50]

[0418] [Table 51]

[0419] Example 16 This example provides an overview of the reactivities of the TCRs of Examples 1-15.

[0420] The TCRs in Table 52 were isolated, expressed in T cells and tested against the relevant antigens. A summary of the results is shown in Table 52.

[0421] [Table 52]

[0422] All references cited in this specification, including publications, patent applications, and patents, are hereby incorporated by reference as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0423] In connection with the description of the present invention (and particularly in connection with the claims which follow), 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 indicated herein or clearly contradicted by context. The use of "at least one of" (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 specified in the specification or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are used in particular Unless otherwise stated, the terms should be construed as open-ended terms (i.e., meaning "including, but not limited to"). The recitation of ranges of values ​​herein is intended to serve merely as a shorthand for individually referring to each separate value within the range, and each separate value is incorporated into the specification as if it were individually recited herein, unless otherwise specified herein. All methods described herein can be performed in any suitable order, unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention, and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0424] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect that such variations will be utilized by those of ordinary 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 clearly contradicted by context.

Claims

1. An isolated or purified T cell receptor (TCR) having antigen specificity for mutant human p53.

2. The TCR described in claim 1 has antigen specificity for human p53 having a mutation at position 175, 220, 245, or 248 of SEQ ID NO:

1.

3. The TCR of claim 1, having antigen specificity for human p53 having one of the following human p53 mutations: R175H, Y220C, G245S, R248Q, or R248W.

4. (1) all of SEQ ID NOs: 27 to 32; (2) all of SEQ ID NOs: 37 to 42; (3) all of SEQ ID NOs: 47 to 52; (4) all of SEQ ID NOs: 57 to 62; (5) all of SEQ ID NOs: 67 to 72; (6) all of SEQ ID NOs: 77 to 82; (7) all of SEQ ID NOs: 87 to 92; (8) all of SEQ ID NOs: 97 to 102; (9) all of SEQ ID NOs: 107 to 112; (10) all of SEQ ID NOs: 117 to 122; (11) all of SEQ ID NOs: 127 to 132; (12) all of SEQ ID NOs: 137 to 142; (13) all of SEQ ID NOs: 147 to 152; (14) all of SEQ ID NOs: 157 to 162; (15) all of SEQ ID NOs: 167 to 172; (16) all of SEQ ID NOs: 177 to 182; (17) all of SEQ ID NOs: 187 to 192; (18) all of SEQ ID NOs: 197 to 202; (19) all of SEQ ID NOs: 207 to 212; (20) all of SEQ ID NOs: 217 to 222; (21) all of SEQ ID NOs: 227 to 232; (22) all of SEQ ID NOs: 237 to 242; (23) all of SEQ ID NOs: 247 to 252; (24) all of SEQ ID NOs: 257 to 262; (25) all of SEQ ID NOs: 267 to 272; (26) all of SEQ ID NOs: 277 to 282; (27) all of SEQ ID NOs: 287 to 292; (28) all of SEQ ID NOs: 297 to 302; (29) all of SEQ ID NOs: 307 to 312; (30) all of SEQ ID NOs: 317 to 322; (31) all of SEQ ID NOs: 327 to 332; (32) all of SEQ ID NOs: 337 to 342; (33) all of SEQ ID NOs: 347 to 352; (34) all of SEQ ID NOs: 357 to 362; (35) all of SEQ ID NOs: 367 to 372; (36) all of SEQ ID NOs: 377 to 382; (37) all of SEQ ID NOs: 387 to 392; (38) all of SEQ ID NOs: 397 to 402; (39) all of SEQ ID NOs: 407 to 412; (40) all of SEQ ID NOs: 417 to 422; (41) all of SEQ ID NOs: 427 to 432; (42) all of SEQ ID NOs: 437 to 442; (43) all of SEQ ID NOs: 447 to 452; (44) all of SEQ ID NOs: 457 to 462; (45) all of SEQ ID NOs: 467 to 472; (46) all of SEQ ID NOs: 477 to 482; or (47) all of SEQ ID NOs: 487 to 492 The TCR according to any one of claims 1 to 3, comprising an amino acid sequence of:

5. (1) both of SEQ ID NOs: 33-34; (2) both of SEQ ID NOs: 43-44; (3) both of SEQ ID NOs: 53-54; (4) both of SEQ ID NOs: 63-64; (5) both of SEQ ID NOs: 73-74; (6) both of SEQ ID NOs: 83-84; (7) both of SEQ ID NOs: 93-94; (8) both of SEQ ID NOs: 103-104; (9) both of SEQ ID NOs: 113-114; (10) both of SEQ ID NOs: 123-124; (11) both of SEQ ID NOs: 133-134; (12) both of SEQ ID NOs: 143-144; (13) both of SEQ ID NOs: 153-154; (14) both of SEQ ID NOs: 163-164; (15) both of SEQ ID NOs: 173-174; (16) both of SEQ ID NOs: 183-184; (17) both of SEQ ID NOs: 193-194; (18) both of SEQ ID NOs: 203-204; (19) both of SEQ ID NOs: 213-214; (20) both of SEQ ID NOs: 223-224; (21) both of SEQ ID NOs: 233 to 234; (22) both of SEQ ID NOs: 243 to 244; (23) both of SEQ ID NOs: 253 to 254; (24) both of SEQ ID NOs: 263 to 264; (25) both of SEQ ID NOs: 273 to 274; (26) both of SEQ ID NOs: 283 to 284; (27) both of SEQ ID NOs: 293 to 294; (28) both of SEQ ID NOs: 303-304; (29) both of SEQ ID NOs: 313 to 314; (30) both of SEQ ID NOs: 323-324; (31) both of SEQ ID NOs: 333 to 334; (32) both of SEQ ID NOs: 343 to 344; (33) both of SEQ ID NOs: 353 to 354; (34) both of SEQ ID NOs: 363 to 364; (35) both of SEQ ID NOs: 373 to 374; (36) both of SEQ ID NOs: 383 to 384; (37) both of SEQ ID NOs: 393 to 394; (38) both of SEQ ID NOs: 403-404; (39) both of SEQ ID NOs: 413 to 414; (40) both of SEQ ID NOs: 423 to 424; (41) both of SEQ ID NOs: 433 to 434; (42) both of SEQ ID NOs: 443 to 444; (43) both of SEQ ID NOs: 453 to 454; (44) both of SEQ ID NOs: 463 to 464; (45) both of SEQ ID NOs: 473 to 474; (46) both of SEQ ID NOs: 483 to 484; or (47) both of SEQ ID NOs: 493 to 494 The TCR according to any one of claims 1 to 4, comprising an amino acid sequence of:

6. (1) both of SEQ ID NOs: 35-36; (2) both of SEQ ID NOs: 45-46; (3) both of SEQ ID NOs: 55-56; (4) both of SEQ ID NOs: 65-66; (5) both of SEQ ID NOs: 75-76; (6) both of SEQ ID NOs: 85-86; (7) both of SEQ ID NOs: 95-96; (8) both of SEQ ID NOs: 105-106; (9) both of SEQ ID NOs: 115-116; (10) both of SEQ ID NOs: 125-126; (11) both of SEQ ID NOs: 135-136; (12) both of SEQ ID NOs: 145-146; (13) both of SEQ ID NOs: 155-156; (14) both of SEQ ID NOs: 165-166; (15) both of SEQ ID NOs: 175-176; (16) both of SEQ ID NOs: 185 to 186; (17) both of SEQ ID NOs: 195-196; (18) both of SEQ ID NOs: 205-206; (19) both of SEQ ID NOs: 215-216; (20) both of SEQ ID NOs: 225-226; (21) both of SEQ ID NOs: 235 to 236; (22) both of SEQ ID NOs: 245 to 246; (23) both of SEQ ID NOs: 255 to 256; (24) both of SEQ ID NOs: 265 to 266; (25) both of SEQ ID NOs: 275 to 276; (26) both of SEQ ID NOs: 285 to 286; (27) both of SEQ ID NOs: 295 to 296; (28) both of SEQ ID NOs: 305-306; (29) both of SEQ ID NOs: 315 to 316; (30) both of SEQ ID NOs: 325 to 326; (31) both of SEQ ID NOs: 335 to 336; (32) both of SEQ ID NOs: 345 to 346; (33) both of SEQ ID NOs: 355 to 356; (34) both of SEQ ID NOs: 365 to 366; (35) both of SEQ ID NOs: 375 to 376; (36) both of SEQ ID NOs: 385 to 386; (37) both of SEQ ID NOs: 395 to 396; (38) both of SEQ ID NOs: 405-406; (39) both of SEQ ID NOs: 415 to 416; (40) both of SEQ ID NOs: 425 to 426; (41) both of SEQ ID NOs: 435 to 436; (42) both of SEQ ID NOs: 445 to 446; (43) both of SEQ ID NOs: 455 to 456; (44) both of SEQ ID NOs: 465 to 466; (45) both of SEQ ID NOs: 475 to 476; (46) both of SEQ ID NOs: 485 to 486; or (47) both of SEQ ID NOs: 495 to 496 The TCR according to any one of claims 1 to 5, comprising an amino acid sequence of:

7. An isolated or purified polypeptide comprising a functional portion of a TCR according to any one of claims 1 to 6, (1) all of SEQ ID NOs: 27 to 32; (2) all of SEQ ID NOs: 37 to 42; (3) all of SEQ ID NOs: 47 to 52; (4) all of SEQ ID NOs: 57 to 62; (5) all of SEQ ID NOs: 67 to 72; (6) all of SEQ ID NOs: 77 to 82; (7) all of SEQ ID NOs: 87 to 92; (8) all of SEQ ID NOs: 97 to 102; (9) all of SEQ ID NOs: 107 to 112; (10) all of SEQ ID NOs: 117 to 122; (11) all of SEQ ID NOs: 127 to 132; (12) all of SEQ ID NOs: 137 to 142; (13) all of SEQ ID NOs: 147 to 152; (14) all of SEQ ID NOs: 157 to 162; (15) all of SEQ ID NOs: 167 to 172; (16) all of SEQ ID NOs: 177 to 182; (17) all of SEQ ID NOs: 187 to 192; (18) all of SEQ ID NOs: 197 to 202; (19) all of SEQ ID NOs: 207 to 212; (20) all of SEQ ID NOs: 217 to 222; (21) all of SEQ ID NOs: 227 to 232; (22) all of SEQ ID NOs: 237 to 242; (23) all of SEQ ID NOs: 247 to 252; (24) all of SEQ ID NOs: 257 to 262; (25) all of SEQ ID NOs: 267 to 272; (26) all of SEQ ID NOs: 277 to 282; (27) all of SEQ ID NOs: 287 to 292; (28) all of SEQ ID NOs: 297 to 302; (29) all of SEQ ID NOs: 307 to 312; (30) all of SEQ ID NOs: 317 to 322; (31) all of SEQ ID NOs: 327 to 332; (32) all of SEQ ID NOs: 337 to 342; (33) all of SEQ ID NOs: 347 to 352; (34) all of SEQ ID NOs: 357 to 362; (35) all of SEQ ID NOs: 367 to 372; (36) all of SEQ ID NOs: 377 to 382; (37) all of SEQ ID NOs: 387 to 392; (38) all of SEQ ID NOs: 397 to 402; (39) all of SEQ ID NOs: 407 to 412; (40) all of SEQ ID NOs: 417 to 422; (41) all of SEQ ID NOs: 427 to 432; (42) all of SEQ ID NOs: 437 to 442; (43) all of SEQ ID NOs: 447 to 452; (44) all of SEQ ID NOs: 457 to 462; (45) all of SEQ ID NOs: 467 to 472; (46) all of SEQ ID NOs: 477 to 482; or (47) all of SEQ ID NOs: 487 to 492 A polypeptide comprising the amino acid sequence of

8. (1) both of SEQ ID NOs: 33-34; (2) both of SEQ ID NOs: 43-44; (3) both of SEQ ID NOs: 53-54; (4) both of SEQ ID NOs: 63-64; (5) both of SEQ ID NOs: 73-74; (6) both of SEQ ID NOs: 83-84; (7) both of SEQ ID NOs: 93-94; (8) both of SEQ ID NOs: 103-104; (9) both of SEQ ID NOs: 113-114; (10) both of SEQ ID NOs: 123-124; (11) both of SEQ ID NOs: 133-134; (12) both of SEQ ID NOs: 143-144; (13) both of SEQ ID NOs: 153-154; (14) both of SEQ ID NOs: 163-164; (15) both of SEQ ID NOs: 173-174; (16) both of SEQ ID NOs: 183-184; (17) both of SEQ ID NOs: 193-194; (18) both of SEQ ID NOs: 203-204; (19) both of SEQ ID NOs: 213-214; (20) both of SEQ ID NOs: 223-224; (21) both of SEQ ID NOs: 233 to 234; (22) both of SEQ ID NOs: 243 to 244; (23) both of SEQ ID NOs: 253 to 254; (24) both of SEQ ID NOs: 263 to 264; (25) both of SEQ ID NOs: 273 to 274; (26) both of SEQ ID NOs: 283 to 284; (27) both of SEQ ID NOs: 293 to 294; (28) both of SEQ ID NOs: 303-304; (29) both of SEQ ID NOs: 313 to 314; (30) both of SEQ ID NOs: 323-324; (31) both of SEQ ID NOs: 333 to 334; (32) both of SEQ ID NOs: 343 to 344; (33) both of SEQ ID NOs: 353 to 354; (34) both of SEQ ID NOs: 363 to 364; (35) both of SEQ ID NOs: 373 to 374; (36) both of SEQ ID NOs: 383 to 384; (37) both of SEQ ID NOs: 393 to 394; (38) both of SEQ ID NOs: 403-404; (39) both of SEQ ID NOs: 413 to 414; (40) both of SEQ ID NOs: 423 to 424; (41) both of SEQ ID NOs: 433 to 434; (42) both of SEQ ID NOs: 443 to 444; (43) both of SEQ ID NOs: 453 to 454; (44) both of SEQ ID NOs: 463 to 464; (45) both of SEQ ID NOs: 473 to 474; (46) both of SEQ ID NOs: 483 to 484; or (47) both of SEQ ID NOs: 493 to 494 The polypeptide of claim 7, comprising the amino acid sequence:

9. (1) both of SEQ ID NOs: 35-36; (2) both of SEQ ID NOs: 45-46; (3) both of SEQ ID NOs: 55-56; (4) both of SEQ ID NOs: 65-66; (5) both of SEQ ID NOs: 75-76; (6) both of SEQ ID NOs: 85-86; (7) both of SEQ ID NOs: 95-96; (8) both of SEQ ID NOs: 105-106; (9) both of SEQ ID NOs: 115-116; (10) both of SEQ ID NOs: 125-126; (11) both of SEQ ID NOs: 135-136; (12) both of SEQ ID NOs: 145-146; (13) both of SEQ ID NOs: 155-156; (14) both of SEQ ID NOs: 165-166; (15) both of SEQ ID NOs: 175-176; (16) both of SEQ ID NOs: 185 to 186; (17) both of SEQ ID NOs: 195-196; (18) both of SEQ ID NOs: 205-206; (19) both of SEQ ID NOs: 215-216; (20) both of SEQ ID NOs: 225-226; (21) both of SEQ ID NOs: 235 to 236; (22) both of SEQ ID NOs: 245 to 246; (23) both of SEQ ID NOs: 255 to 256; (24) both of SEQ ID NOs: 265 to 266; (25) both of SEQ ID NOs: 275 to 276; (26) both of SEQ ID NOs: 285 to 286; (27) both of SEQ ID NOs: 295 to 296; (28) both of SEQ ID NOs: 305-306; (29) both of SEQ ID NOs: 315 to 316; (30) both of SEQ ID NOs: 325 to 326; (31) both of SEQ ID NOs: 335 to 336; (32) both of SEQ ID NOs: 345 to 346; (33) both of SEQ ID NOs: 355 to 356; (34) both of SEQ ID NOs: 365 to 366; (35) both of SEQ ID NOs: 375 to 376; (36) both of SEQ ID NOs: 385 to 386; (37) both of SEQ ID NOs: 395 to 396; (38) both of SEQ ID NOs: 405-406; (39) both of SEQ ID NOs: 415 to 416; (40) both of SEQ ID NOs: 425 to 426; (41) both of SEQ ID NOs: 435 to 436; (42) both of SEQ ID NOs: 445 to 446; (43) both of SEQ ID NOs: 455 to 456; (44) both of SEQ ID NOs: 465 to 466; (45) both of SEQ ID NOs: 475 to 476; (46) both of SEQ ID NOs: 485 to 486; or (47) both of SEQ ID NOs: 495 to 496; 9. The polypeptide of claim 7 or 8, comprising the amino acid sequence:

10. (1) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 27-29 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 30-32; (2) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 37-39 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 40-42; (3) 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; (4) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 57-59 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 60-62; (5) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 67-69 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 70-72; (6) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 77-79 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 80-82; (7) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 87-89 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 90-92; (8) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 97-99 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 100-102; (9) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 107-109 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 110-112; (10) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 117-119 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 120-122; (11) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 127-129 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 130-132; (12) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 137-139 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 140-142; (13) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 147-149 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 150-152; (14) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 157-159 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 160-162; (15) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 167-169 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 170-172; (16) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 177-179 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 180-182; (17) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 187-189 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 190-192; (18) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 197-199 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 200-202; (19) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 207-209 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 210-212; (20) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 217-219 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 220-222; (21) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 227-229 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 230-232; (22) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 237-239 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 240-242; (23) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 247-249 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 250-252; (24) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 257-259 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 260-262; (25) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 267-269 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 270-272; (26) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 277-279 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 280-282; (27) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 287-289 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 290-292; (28) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 297-299 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 300-302; (29) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 307-309 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 310-312; (30) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 317-319 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 320-322; (31) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 327-329 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 330-332; (32) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 337-339 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 340-342; (33) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 347-349 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 350-352; (34) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 357-359 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 360-362; (35) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 367-369 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 370-372; (36) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 377-379 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 380-382; (37) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 387-389 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 390-392; (38) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 397-399 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 400-402; (39) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 407-409 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 410-412; (40) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 417-419 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 420-422; (41) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 427-429 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 430-432; (42) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 437-439 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 440-442; (43) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 447-449 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 450-452; (44) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 457-459 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 460-462; (45) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 467-469 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 470-472; (46) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 477-479 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 480-482; or (47) A first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 487 to 489 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 490 to 492. An isolated or purified protein comprising:

11. (1) 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; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:43 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:44; (3) 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; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:63 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:64; (5) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:73 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:74; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 83 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 84; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:93 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:94; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 103 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 104; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 113 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 114; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 123 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 124; (11) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 133 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 134; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 143 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 144; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 153 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 154; (14) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 163 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 164; (15) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 173 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 174; (16) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 183 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 184; (17) 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: 194; (18) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 203 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 204; (19) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:213 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:214; (20) 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: 224; (21) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 233 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 234; (22) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 243 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 244; (23) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 253 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 254; (24) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 263 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 264; (25) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:273 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:274; (26) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 283 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 284; (27) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:293 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:294; (28) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 303 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 304; (29) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 313 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 314; (30) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 323 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 324; (31) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 333 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 334; (32) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 343 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 344; (33) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 353 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 354; (34) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 363 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 364; (35) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 373 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 374; (36) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 383 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 384; (37) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 393 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 394; (38) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:403 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:404; (39) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:413 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:414; (40) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 423 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 424; (41) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 433 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 434; (42) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 443 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 444; (43) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 453 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 454; (44) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 463 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 464; (45) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:473 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:474; (46) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 483 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 484; or (47) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 493 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:

494. The protein of claim 10 .

12. (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:35 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:36; (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:45 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:46; (3) 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; (4) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:65 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:66; (5) 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; (6) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 85 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 86; (7) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:95 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:96; (8) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 105 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 106; (9) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 115 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 116; (10) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 125 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 126; (11) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 135 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 136; (12) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 145 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 146; (13) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 155 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 156; (14) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 165 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 166; (15) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 175 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 176; (16) 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: 186; (17) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 195 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 196; (18) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 205 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 206; (19) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:215 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:216; (20) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 225 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 226; (21) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 235 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 236; (22) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 245 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 246; (23) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:255 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:256; (24) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:265 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:266; (25) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:275 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:276; (26) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:285 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:286; (27) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:295 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:296; (28) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 305 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 306; (29) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 315 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 316; (30) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 325 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 326; (31) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 335 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 336; (32) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 345 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 346; (33) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 355 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 356; (34) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 365 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 366; (35) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 375 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 376; (36) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 385 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 386; (37) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 395 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 396; (38) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:405 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:406; (39) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:415 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:416; (40) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 425 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 426; (41) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 435 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 436; (42) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 445 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 446; (43) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:455 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:456; (44) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 465 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 466; (45) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO:475 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:476; (46) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 485 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 486; or (47) A first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 495 and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:

496. The protein of claim 10 or 11, comprising:

13. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 6, a polypeptide according to any one of claims 7 to 9, or a protein according to any one of claims 10 to 12.

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

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

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

17. 17. A pharmaceutical composition comprising: (a) a TCR according to any one of claims 1 to 6, a polypeptide according to any one of claims 7 to 9, a protein according to any one of claims 10 to 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, or a population of cells according to claim 16; and (b) a pharma- ceutical acceptable carrier.

18. 1. A method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising cells of said cancer with a TCR according to any one of claims 1 to 6, a polypeptide according to any one of claims 7 to 9, a protein according to any one of claims 10 to 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, a population of cells according to claim 16, or a pharmaceutical composition according to claim 17, thereby forming a complex; (b) detecting the complex; and Including, Detection of said complex indicates the presence of cancer in said mammal.

19. 18. A TCR according to any one of claims 1 to 6, a polypeptide according to any one of claims 7 to 9, a protein according to any one of claims 10 to 12, a nucleic acid according to claim 13, a recombinant expression vector according to claim 14, a host cell according to claim 15, a population of cells according to claim 16, or a pharmaceutical composition according to claim 17, for use in the treatment or prevention of cancer in a mammal.

20. 20. A population of cells for use according to claim 19 which is autologous to the mammal.

21. 20. A population of cells for use according to claim 19 which is allogeneic to said mammal.

22. 22. The TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use according to the method of claim 18 or any one of claims 19 to 21, wherein the cancer is an epithelial cancer.

23. 22. The method of claim 18, or the TCR, polypeptide, protein, nucleic acid, recombinant expression vector, host cell, population of cells, or pharmaceutical composition for use according to any one of claims 19 to 21, 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.

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