T cell receptor that recognizes mutant 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 September 29, 2017, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Project No. BC010985 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, filed concurrently herewith and identified as follows, is incorporated herein by reference in its entirety: a 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 treatments such as surgery, chemotherapy, and radiation therapy, the prognosis for many cancers, such as pancreatic, colorectal, lung, endometrial, ovarian, and prostate cancers, may be poor. Thus, there is an unmet need for additional cancer treatments. Summary of the Invention

[0005] An embodiment of the present invention provides an isolated or purified T cell receptor (TCR) with antigen 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 explanation of the drawings]

[0008] [Figure 1] Graph showing the concentration of IFN-γ (pg / mL) measured after co-culture of autologous PBLs transduced with one of the indicated TCRs from patient 4127 with autologous dendritic cells (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). [Figure 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. [Figure 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 with autologous PBLs transduced with one of the indicated TCRs from patient 4127. [Figure 4] Graph showing the 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. [Figure 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 TILs from patient 4127 with allogeneic (DRB3*01:01:01 or DRB3*02:02:01) APCs electroporated with TMG composed of irrelevant (IRR; left hatched white bar), WT p53 (p53-WT; left hatched gray bar), or mutant p53 (p53-MUT; gray bar) sequences, or pulsed with (2) peptide vehicle (DMSO; right hatched white bar) or purified (>95% by HPLC) 25-amino acid peptide composed of the WT p53-G245 sequence (LP-p53-wt-G245; right hatched gray bar) or mutant p53-G245S (LP-p53-mut-G245S; black bar) sequences. [Figure 7] (1) TMG composed of irrelevant (IRR; left hatched white bar), WT p53 (p53-WT; left hatched gray bar), or mutant p53 (p53-MUT; gray bar) sequences was electroporated, or (2) peptide vehicle (DMSO; right hatched white bar), or WT p53-G245 sequence (LP-p53-wt-G245; right hatched gray bar) or mutant p53 (p53-MUT; gray bar) sequences were electroporated. Figure 11 is a graph showing the number of spots per 2 x 10 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 / 2 x 10 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 / 2 x 10 cells) measured after co-culture of autologous DCs pulsed with the candidate minimal epitope (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 a control. [Figure 9] Figures 9A-9D are graphs showing the percentage of mTCRβ CD8+41BB+ cells detected after coculture 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. Coculture of pulsed DCs with untransduced (UT) cells (triangles) served as a control. Results shown are representative of two experiments. [Figure 10] Figures 10A-10B are graphs showing IFN-γ secretion (pg / mL) (A) and the percentage of 4-1BB-positive cells (%, mTCRβ+) (B) measured after coculture of T2 cells pulsed with the indicated concentrations of minimally mutated epitopes with cells transduced with one of the following TCRs: AV12 / BV6-1 (black triangles), AV6 / BV11 (circles), or AV38 / BV10-3 (squares). Coculture 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 2 × 10 effector cells detected after coculture 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 (-). Untransduced effector cells (unshaded bars) cocultured with the target cell lines were used as a control. [Figure 12] Graph showing the number of IFN-γ-positive spots per 2 × 10 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 × 10 effector cells measured after co-culture of TIL fragments F1 to F24 (n = 24) from patient 4238 with autologous APCs pulsed with either 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 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 to 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 coculture of TIL fragments (F1–F24; n = 24) derived from patient 4253 with autologous APCs pulsed with (1) peptide vehicle (DMSO; white bars), pulsed with (2) a purified (>95% by HPLC) 25-amino acid peptide composed of the mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence, electroporated with (3) an irrelevant TMG (TMG-IRR; gray hatched bars), or electroporated with p53-mut-TMG containing the mutant p53-R248W sequence. [Figure 17] Graph showing the number of IFN-γ-positive spots per 2 × 10 effector cells measured after coculture of TIL fragments (F1–F24, n = 24) derived from patient 4273 with autologous APCs electroporated with TMGs (TMGs) composed of irrelevant (IRR; white bars), WT p53 (p53-WT; gray bars), or mutant p53 (p53-MUT; black bars) sequences. [Figure 18] Graph showing the percentage of CD4+4-1BB+ cells detected after coculture of TIL fragments F1 to F24 (n=24) from patient 4273 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 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 APCs 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; gray 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 APCs 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; gray 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 with 4149-TCRa2b1 or 4149-TCRa2b2) with autologous APCs (1) electroporated with TMG composed of irrelevant (IRR; black bars with right diagonal lines), WT p53 (p53-wt-TMG; black bars with left diagonal lines), 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 lines) or mutant p53-Y220C (LP-p53-Y220C-MUT; black bars). Phorbol 12-myristate 13-acetate (PMA) and ionomycin (Iono) were positive controls (gray bars). [Figure 22]Graph showing the percentage of CD4+4-1BB+ cells detected after co-culture of autologous PBLs from patient 4149 transfected with TCR (4149-TCRa2b1 or 4149-TCRa2b2) with autologous APCs (1) electroporated with TMG composed of irrelevant (IRR; black bars with right hatched lines), WT p53 (p53-wt-TMG; black bars with left hatched lines), 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 lines) or mutant p53-Y220C (LP-p53-Y220C-MUT; black bars). PMA and Iono were positive controls (gray 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] Figure 1 shows graphs of IFN-γ secretion (pg / mL) after co-culture of Cos7 cells co-transfected with individual HLA alleles + / - TMG with 4149-TCRa2b2 transferred T cells. Non-TMG-transfected cells were pulsed with the p53Y220C 15-mer peptide. Pulsed target cells are indicated by shaded bars. TMG-transfected target cells are indicated by unshaded bars. [Figure 25] Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments (F2 and F24) 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 2×10 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 the 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 × 10 effector cells measured after coculture of TIL fragments (F1–F24, n = 24) derived from patient 4268 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 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) from patient 4268 with autologous APCs 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; gray 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) from patient 4268 with autologous APCs 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; gray bars) or mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Figure 30]Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments (F1-F24, n=24) from patient 4268 with autologous APCs 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; gray bars) or mutant p53-R248Q (LP-p53-R248Q-MUT; black bars) sequence. [Figure 31] Graph showing the number of IFN-γ-positive spots per 2 × 10 effector cells measured after coculture of TIL fragments (F1–F24, n = 24) derived from patient 4266 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 32] Graph showing the percentage of CD8+4-1BB+ cells detected after coculture of TIL fragments (F1 to F24, n=24) derived from patient 4266 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 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) from patient 4266 with autologous APCs 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; gray bars) or mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence. [Figure 34]Graph showing the percentage of CD8+4-1BB+ cells detected after co-culture of TIL fragments (F1-F24, n=24) from patient 4266 with autologous APCs 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; gray bars) or mutant p53-R248W (LP-p53-R248W-MUT; black bars) sequence. [Figure 35] Alignment of the amino acid sequences of nine p53 splice variants is shown: 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). [Figure 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 (% 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 × 10 cells) (left y-axis; hatched bars) measured after coculture of TILs (fragment culture 12) from patient 4141 with autologous APCs transfected with TMG encoding an irrelevant mutation (TMG-IRR), the WT p53 sequence (TP53-wt-TMG), or a mutant p53 sequence containing R175H (TP53-mut-TMG). Medium alone, and PMA and ionomycin served as negative and positive controls, respectively. [Figure 39] Graph showing the number of IFN-γ-positive spots per 2 × 10 effector cells measured after coculture of TILs from patient 4141 (fragment culture 12) with Cos7 cells cotransfected with the indicated HLA allele and no additional genes (HLA only; white bars), WT TP53 TMG (gray hatched bars), or mutant TP53 TMG containing the p53-R175H sequence (black bars). [Figure 40] This graph shows the concentration of IFN-γ (pg / mL) measured after co-culture of mock (no TCR) or 4141-TCR1a2-expressing T cells with T2 tumor cells (expressing HLA-A*02:01). T2 cells were pulsed with peptide vehicle (DMSO; gray bars) or purified (>95% by HPLC) peptides consisting of WT p53-R175 peptide (hatched gray bars) or mutant p53-R175H peptide (black bars). Medium alone (white bars) and PMA and ionomycin (checkered bars) served as negative and positive controls, respectively. Data are mean ± SEM (n=3). [Figure 41]Figure 1 shows 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 unengineered (unshaded bars) or overexpressed the full-length p53-R175H protein (shaded bars) with T cells expressing 4141-TCR1a2. Data are mean ± SEM (n = 3). For statistical analysis, a two-tailed Student's t-test was performed for each cytokine between the two cell lines (***p < 0.001). [Figure 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) sequences. [Figure 43] Graph showing the percentage of CD4+4-1BB positive cells detected after co-culture of TIL fragment cultures (#6) derived from patient 4259 with autologous APCs pulsed for 2 hours at 37°C with decreasing concentrations of a 25 amino acid peptide corresponding to the WT p53 sequence (white circles) or mutant p53-Y220C (black squares). [Figure 44] 1 is a graph showing the percentage of CD4+4-1BB positive cells detected after co-culture of TILs derived from patient 4259 with autologous APCs pulsed with DMSO, WT p53-Y220 peptide, or mutant p53-Y220C peptide. [Figure 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). [Figure 46] This graph shows the percentage of 4-1BB+ cells detected after coculture of effector T cells (10 cells) 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 either W6 / 32 pan-HLA class I-specific blocking antibody, IVA12 pan-HLA class II-specific blocking antibody, or mutant p53-Y220C peptide. Medium alone (no TCR), PMA, and ionomycin served as negative and positive controls, respectively. Data are mean ± SEM (n=3). For statistical analysis, a 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 × 10 effector cells measured after coculture 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 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 APCs 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; gray 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 coculture of TIL fragments (F1 to 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 APCs 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; gray 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 coculture 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 APCs 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 coculture of TILs from patient 4266 with Cos7 cells cotransfected with individual HLA alleles from patient 4266 and pulsed with no peptide (white bars), DMSO (peptide vehicle; gray bars), wild-type p53-R248 peptide (gray shaded bars), or mutant p53-R248W peptide (black bars). Data are mean ± SEM (n=3). [Figure 54] Figure 1 shows the percentage of 4-1BB+ cells (% of CD8+) detected after co-culture of T cells expressing mock (no TCR), 4266-TCR1, 4266-TCR2, 4266-TCR3, or 4266-TCR4 with autologous APCs pulsed with peptide vehicle (DMSO; gray bars) or purified (>95% by HPLC) 25-amino acid peptides composed of the WT p53-R248 sequence (hatched gray bars) or mutant p53-R248W (black bars). Medium alone (white bars) and PMA and ionomycin (checkered bars) served as negative and positive controls, respectively. [Figure 55]Figure 1 shows the percentage of 4-1BB+ cells (% of CD8+) detected after coculture 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 T cells expressing mock (no TCR) or p53-R248W-specific TCRs (4266-TCR2, 4266-TCR3, or 4266-TCR4). TC#4266 cells were incubated with either the W6 / 32 pan-HLA class I-specific blocking antibody (right gray hatched bar), the IVA12 pan-HLA class II-specific blocking antibody (gray bar), or the mutant p53-R248W peptide (left gray hatched bar). 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 coculture of autologous APCs transfected with TMG encoding an unrelated 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 (open circles) or mutant (closed squares) derived from the p53-R248W neoepitope with TILs from patient 4273. DMSO was the peptide vehicle. [Figure 58]This graph shows the percentage of CD4+4-1BB+ cells detected after coculture of autologous APCs pulsed with a 15-amino acid peptide (thickened amino acid substitutions) derived from the p53-R248W neoepitope, which overlaps 14 amino acids, and TILs from patient 4273. 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) served as additional controls for the 15-amino acid peptide. The peptides are YMCNSSCMGGMNWRP (SEQ ID NO: 592); MCNSSCMGGMNWRPIL (SEQ ID NO: 593); CNSSCMGGMNWRPIL (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 the 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 the p53-R248W neoantigen, respectively. [Figure 60] Figure 1 shows the number of IFN-γ spots per 2 × 10 cells measured after co-culture of mock (no TCR) or 4273-TCR1a2-expressing T cells with autologous APCs pulsed with peptide vehicle (DMSO; gray bars) or purified (>95% by HPLC) 25-amino acid peptides composed of the WT p53-R248 sequence (hatched gray bars) or mutant p53-R248W (black bars) sequences. Medium alone (white bars) and PMA and ionomycin (checkered bars) served as negative and positive controls, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0009] The tumor protein P53 (also referred to as "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 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 prevents the cell from dividing and signals the cell to undergo apoptosis. By stopping the division of cells with mutant or damaged DNA, p53 helps prevent tumor development. Wild-type (WT) (normal) full-length p53 comprises the amino acid sequence of SEQ ID NO: 1.

[0010] Mutations in the p53 protein can reduce or eliminate the tumor suppressor function of the p53 protein. Alternatively, or in addition, the p53 mutation can be a gain-of-function mutation by interfering with WT p53 in a dominant-negative manner. Mutant p53 proteins can 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] Embodiments of the present invention provide isolated or purified T cell receptors (TCRs) having antigen specificity for mutant human p53 (hereinafter "mutant p53"). Hereinafter, reference to "TCR" also refers to functional portions and functional variants of the TCR, unless otherwise specified. p53 mutations are defined herein by reference to the amino acid sequence of full-length WT p53 (SEQ ID NO: 1). Accordingly, p53 mutations are described herein by reference to the amino acid residue present at a particular position, followed by the position number, followed by the amino acid that replaces that residue in the particular mutation under consideration. 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 Figure 35. Thus, T cells isolated by the methods of the present invention can have antigen specificity for any mutant p53 amino acid sequence described herein encoded by any of the nine p53 splice variants. Where 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 corresponding to the specified position in the 393-amino acid sequence of SEQ ID NO: 1, with the understanding that the actual position in the splice variant may differ.

[0012] In an embodiment of the present 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 embodiments 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 may be capable of recognizing mutant p53 in an HLA (human leukocyte antigen) molecule-dependent manner. As used herein, "HLA molecule-dependent manner" means that the TCR elicits an immune response when it binds to mutant p53 within the framework of HLA molecules expressed in the patient from which the TCR was isolated. The TCR of the present invention may be capable of recognizing mutant p53 presented by applicable HLA molecules and may 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, can provide many advantages. Mutant p53 is expressed in cancer cells but not in normal, non-cancerous cells. Without being bound by any 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-cancerous cells, e.g., by minimizing or eliminating toxicity. Furthermore, the TCRs of the present invention can advantageously successfully treat or prevent mutant p53-positive cancers that do not respond to other types of treatment, such as chemotherapy, surgery, or radiation. Furthermore, the TCRs of the present invention can 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 harboring p53 mutations, or a combination thereof). Approximately half of all tumors harbor mutations in p53, about half of which are missense mutations, with approximately 30% of these missense mutations occurring at the following "hotspot" residues: R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273C, R273L, R273H, and R282W. Furthermore, the same "hotspot" mutations in p53 (e.g., R175H, Y220C, G245D, G245S, R248L, R248Q, R248W, R249S, R273H, R273C, R273L, or R282W) frequently occur in unrelated human tumors (cumulatively accounting for approximately 30% of p53 missense mutations). Thus, the TCRs of the present invention may increase the number of patients potentially eligible for treatment with immunotherapy.

[0015] The term "antigen specificity" as used herein means that a 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 so that the target cells express mutant p53. Approximately 1 x 10 TCR-expressing cells were co-cultured with antigen-negative, HLA-positive target cells transfected with a nucleotide sequence encoding 3. 4 ~Approx. 1×10 5 When a T cell secretes IFN-γ of 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 are also able to 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 so 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., derived from PBMCs that do not express 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 an IFN-γ-secreting TCR is at least twice as high as the number of IFN-γ-secreting negative control T cells when cocultured 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 transfected with a nucleotide sequence encoding mutant p53 so that the target cells express mutant p53. The peptide concentration and negative control may be as described herein for other aspects of the invention. The number of IFN-γ-secreting cells can be measured by methods known in the art, such as enzyme-linked immunosorbent spot (ELISPOT) assays.

[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 transfected with a nucleotide sequence encoding mutant p53 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. 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 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 so that the target cells express mutant p53. The concentration of peptide 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 the 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 target cells expressing mutant p53.

[0021] The present invention provides TCRs comprising two polypeptides (i.e., polypeptide chains), such as 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 TCR polypeptides of the present invention may comprise any amino acid sequence, as 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 regions (CDRs) 1, 2, and 3. In an embodiment of the invention, a TCR comprises a first polypeptide chain comprising CDR1 of the α chain (CDR1α), CDR2 of the α chain (CDR2α), and CDR3 of the α chain (CDR3α), and a second polypeptide chain comprising CDR1 of the β chain (CDR1β), CDR2 of the β chain (CDR2β), and CDR3 of the β chain (CDR3β).In an embodiment of the present 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, a 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 above collections of CDRs. 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: 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: 233-234; (22) both of SEQ ID NOs: 243-244; (23) both of SEQ ID NOs: 253-254; (24) both of 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 of the collections of 47 amino acid sequences described above in this paragraph represents two variable regions of each of 47 different TCRs with antigen specificity for mutant human p53. The two amino acid sequences in each collection correspond to the variable regions of the α and β chains of the TCRs, respectively.

[0024] The TCRs of the present invention may further comprise a constant region. The constant region may be derived from any suitable species, such as, for example, a human or a mouse. In embodiments of the present invention, the TCR further comprises a mouse constant region. As used herein, the terms "mouse" and "human," when referring to a TCR or any component of a TCR described herein (e.g., complementarity-determining regions (CDRs), variable region, constant region, alpha chain, and / or beta chain), refer to a TCR (or component thereof) of mouse or human origin, respectively, i.e., a TCR (or component thereof) that originates from or was once expressed by a mouse T cell or a human T cell, respectively. In embodiments of the present invention, the TCR may comprise a mouse α chain constant region and a mouse β chain constant region. The mouse α chain constant region may be modified or unmodified. The modified mouse α chain constant region may be, for example, cysteine-substituted, LVL-modified, or both cysteine-substituted and LVL-modified, e.g., as described in U.S. 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 U.S. Patent Application Publication No. 2017 / 0145070. In embodiments 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 embodiments 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. This type of 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 present 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 (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) 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 chain and β chain 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] 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, for example, variants of the TCRs, polypeptides, or proteins described herein (parent TCRs, polypeptides, or proteins) that retain, to a similar, identical, or greater extent than the parent TCR, polypeptide, or protein, the antigen specificity of the parent TCR, or the ability to specifically bind to 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 with at least one conservative amino acid substitution. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with particular physical and / or chemical properties is replaced with another amino acid with the same chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), substitutions of an amino acid having a non-polar side chain with an amino acid having a different non-polar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), substitutions of a basic amino acid with another basic amino acid (Lys, Arg, etc.), substitutions of an amino acid having a polar side chain with an amino acid having a different polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

[0028] Alternatively, or in addition, a functional variant may comprise the amino acid sequence of a parent TCR, polypeptide, or protein with at least one non-conservative amino acid substitution. In this case, it is preferred that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conservative amino acid substitution enhances the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent TCR, polypeptide, or protein.

[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 present 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] With respect to the polypeptides of the present 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 reference to a TCR, refers to any portion or fragment of a TCR of the present invention that retains the biological activity of the TCR of which it is a part (the parent TCR). A functional portion includes, for example, a portion of a TCR that retains the ability to specifically bind to mutant p53 or to detect, treat, or prevent cancer to a similar, identical, or greater extent than the parent TCR (e.g., depending on the applicable HLA molecule). With respect to the parent TCR, a functional portion may constitute, for example, 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 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 present 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-mentioned 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: 153 to 154; (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 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; (2) the sequences (3) both of sequence numbers 45 to 46; (4) both of sequence numbers 55 to 56; (5) both of sequence numbers 65 to 66; (6) both of sequence numbers 85 to 86; (7) both of sequence numbers 95 to 96; (8) both of sequence numbers 105 to 106; (9) both of sequence numbers 115 to 116; (10) both of sequence numbers 125 to 126; (11) both of sequence numbers 135 to 136; (12) both of sequence numbers 145 to 146; (13) both of sequence numbers 155 to 156; (14) both of sequence numbers 165 to 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: 275-276; (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 SEQ ID NOs: 415-416; (40) both SEQ ID NOs: 425-426; (41) both SEQ ID NOs: 435-436; (42) both SEQ ID NOs: 445-446; (43) both SEQ ID NOs: 455-456; (44) both SEQ ID NOs: 465-466; (45) both SEQ ID NOs: 475-476; (46) both SEQ ID NOs: 485-486; or (47) both SEQ ID NOs: 495-496.

[0037] The present invention further provides proteins 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; or (4) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 57 to 59. (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 comprising all of the amino acid sequences of SEQ ID NOs: 97 to 99 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 100 to 102; (9) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 107 to 109 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 110 to 112; (10) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 117 to 119 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 120 to 122; (11) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 127 to 129 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 128 to 129; (11) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 130 to 132 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 137 to 139; (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: 150 to 152.(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 comprising all of the amino acid sequences of SEQ ID NOs: 197 to 199 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 200 to 202; (19) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 207 to 209 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 210 to 212; (20) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 217 to 219 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 220 to 222. (21) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 227 to 229 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 230 to 232; (22) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 237 to 239 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 240 to 242; (23) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 247 to 249 and a second polypeptide chain comprising 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; (42) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 437 to 439 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 440 to 442; (43) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 447 to 449 and a second polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 450 to 452; (44) a first polypeptide chain comprising all of the amino acid sequences of SEQ ID NOs: 457 to 459 and a second polypeptide chain comprising 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 present 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 an 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 present 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 (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. (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 (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: 226; (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 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 present invention may be a TCR. Alternatively, the protein of the present invention may be a fusion protein, where the first and / or second polypeptide chain(s) of the protein further comprise another amino acid sequence, e.g., an amino acid sequence encoding an immunoglobulin or a portion thereof. In this regard, the present invention also provides fusion proteins comprising at least one of the polypeptides of the present invention described herein together with at least one other polypeptide. The other polypeptide may be present as a separate protein of the fusion protein, or as a polypeptide expressed in frame (tandem) with one of the polypeptides of the present invention described herein. The other polypeptide may encode any peptidic or proteinaceous molecule, or a portion thereof, including, but not limited to, an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.

[0041] A fusion protein can include one or more copies of a polypeptide of the invention and / or one or more copies of another polypeptide. For example, a fusion protein can include 1, 2, 3, 4, 5, or more copies of a polypeptide of the invention and / or another polypeptide. Suitable methods for making fusion proteins are known in the art and include, for example, recombinant methods.

[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 connecting the α chain and the β chain. 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 TCR, polypeptide, and / or protein in a host cell. The linker peptide may comprise any suitable amino acid sequence. For example, the linker peptide may 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 α and β chains. In embodiments of the invention, the TCR, polypeptide, or protein may comprise an amino acid sequence comprising a full-length α chain, a full-length β chain, and a linker peptide located between the α and β chains.

[0043] The protein of the present invention may be a recombinant antibody or antigen-binding portion thereof comprising at least one of the polypeptides of the present invention described herein. As used herein, "recombinant antibody" refers to a recombinant (e.g., genetically engineered) protein comprising at least one of the polypeptides of the present invention and an antibody polypeptide chain or antigen-binding portion thereof. The antibody polypeptide or antigen-binding portion thereof may be the antibody heavy chain, light chain, variable or constant region of the heavy or light chain, 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 in the 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 present invention. 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 present 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, polypeptides may range in length from about 50 to about 5,000 amino acids, e.g., 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1,000 or more amino acids. In this regard, polypeptides of the present 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, 2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[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, for example, de novo synthesis. Polypeptides and proteins can also be produced recombinantly using standard recombinant methods and the nucleic acids described herein. See, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual ,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] Embodiments of the present invention provide nucleic acids 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." The term "nucleic acid" generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages, instead of the phosphodiester linkages found between nucleotides in unmodified oligonucleotides. In embodiments, nucleic acids include complementary DNA (cDNA). It is generally preferred that nucleic acids do not contain any insertions, deletions, inversions, and / or substitutions. However, in some instances, it may be preferable for nucleic acids to contain one or more insertions, deletions, inversions, and / or substitutions, as discussed herein.

[0049] Preferably, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" refers to (i) a molecule constructed outside a living cell by joining natural or synthetic nucleic acid segments into a nucleic acid molecule capable of replicating in the living cell, or (ii) a molecule obtained by replication of the above (i). For purposes herein, replication may be in vitro or in vivo.

[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 et al., supra. For example, nucleic acids can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides (e.g., phosphorothioate derivatives and acridine-substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed upon hybridization. Examples of modified nucleotides that can be used to generate nucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylquefen, inosine, N-acetyl-3-methyl-4-methyl-5-methyl-1-methyl-2 ... 6 -Isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -substituted adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N 6Examples of nucleic acids include, but are not limited to, -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 embodiments of the present invention, the nucleic acid comprises a codon-optimized nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein. Without being bound by any particular theory or mechanism, it is believed that codon optimization of a nucleotide sequence increases the translation efficiency of an mRNA transcript. Codon optimization of a nucleotide sequence 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. Optimizing 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 nucleic acids 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. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (any nucleotide sequence of a nucleic acid described herein) in an amount detectably greater than nonspecific hybridization. High stringency conditions include conditions that distinguish polynucleotides with exact complementary sequences, or those containing only a few scattered mismatches, from random sequences that happen to have a few small regions (e.g., 3-10 bases) 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 readily distinguishable by high stringency hybridization. Relatively high stringency conditions include low salt and / or high temperature conditions, such as those provided by about 0.02-0.1 M NaCl or equivalent and temperatures of about 50-70°C. Such high stringency conditions tolerate little, if any, mismatch between the nucleotide sequence and the template or target strand and are particularly suitable for detecting expression of any of the TCRs of the present invention. It will generally be appreciated that conditions can be made more stringent by adding increasing amounts of formamide.

[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 can consist essentially of any of the nucleotide sequences described herein.

[0055] The nucleic acids of the present invention can be incorporated into recombinant expression vectors. In this regard, the present invention provides recombinant expression vectors comprising any of the nucleic acids of the present invention. In embodiments of the present invention, the recombinant expression vector comprises nucleotide sequences encoding the α chain, the β chain, and the linker peptide.

[0056] For purposes herein, the term "recombinant expression vector" refers to a genetically engineered oligonucleotide or polynucleotide construct that contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and causes a host cell to express the mRNA, protein, polypeptide, or peptide when the vector is contacted with the cell under conditions sufficient to cause expression of the mRNA, protein, polypeptide, or peptide in the cell. The vectors of the present invention are not naturally occurring in their entirety. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may contain any type of nucleotide, including, but not limited to, DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. The recombinant expression vector may contain naturally occurring internucleotide linkages, non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or modified nucleotides or internucleotide linkages do not interfere with transcription or replication of the vector.

[0057] The recombinant expression vectors 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 plasmids and viruses, for propagation and propagation, or for expression. Examples of suitable vectors include those designed for gene expression, 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 present invention can be prepared using standard recombinant DNA techniques, for example, as described in Green and Sambrook et al., supra. Expression vector constructs, either circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from ColEl, 2μ plasmid, λ, SV40, bovine papilloma virus, etc.

[0059] Desirably, the recombinant expression vector includes regulatory sequences, e.g., transcription and translation initiation and termination codons, as appropriate, and considering whether the vector is DNA- or RNA-based, specific for the type of host cell (e.g., bacterial, fungal, plant, or animal) into which the vector will be introduced.

[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 comprise a native or non-native promoter operably linked to a nucleotide sequence encoding a TCR, polypeptide, or protein, or to a nucleotide sequence complementary to or hybridizing to the 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 in the art. Similarly, the combination of a nucleotide sequence and a promoter is also within the skill of one 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, SV40 promoter, RSV promoter, and promoters 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 also cause the cell in which it is expressed to become resistant to an agent, e.g., a drug. The suicide gene may be a gene that confers sensitivity to an agent and causes the cell to die when the cell comes into contact with or is 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 capable of containing 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 protozoan. The host cell may be a cultured cell or a primary cell, i.e., a cell isolated directly from an organism, such as a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in 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, etc. For the purpose of amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, such as a DH5α cell. For the purpose 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. Although the host cell may be of any cell type, may be derived from any type of tissue, and may be at any stage of development, the host cell is preferably a peripheral blood lymphocyte (PBL) or 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 cell may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell may also be enriched or purified. Preferably, the T cell is a human T cell. The T cell may be any type of T cell, may be at any stage of development, and may be CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD4 + T cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes (TILs), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, and the like.

[0066] Also provided by the present invention are populations of cells comprising at least one host cell described herein. The population of cells may be a heterogeneous population comprising host cells comprising any of the described recombinant expression vectors in addition to at least one other cell, e.g., a host cell (e.g., a T cell), that does not comprise any of the recombinant expression vectors, or cells other than T cells, e.g., B cells, macrophages, neutrophils, erythrocytes, hepatocytes, endothelial cells, epithelial cells, muscle cells, brain cells, etc. Alternatively, the population of cells may be a substantially homogeneous population comprising primarily host cells comprising (e.g., consisting essentially of) the recombinant expression vector. The population may also be a clonal population of cells, in which all cells in the population are clones of a single host cell comprising the recombinant expression vector, such that all cells in the population comprise the recombinant expression vector. In one embodiment of the present invention, the population of cells is a clonal population comprising host cells comprising the recombinant expression vectors described herein.

[0067] In embodiments of the present 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 achieved 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, 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 even about 100%.

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

[0070] Preferably, the carrier is a pharmaceutically acceptable carrier. For pharmaceutical compositions, the carrier may be any of those conventionally used for the particular TCR material of the invention under consideration. Methods of preparing administrable compositions will be known or apparent to those skilled in the art, and are described, for example, in Remington: The Science and Practice of Pharmacy, 22 nd Ed., Pharmaceutical Press (2012). Preferably, a pharmaceutically acceptable carrier is one that has no detrimental side effects or toxicity under the conditions of use.

[0071] The choice of carrier will be determined in part by the specific TCR material of the invention, as well as by the specific method used to administer the TCR material of the invention. Accordingly, there are a variety of suitable formulations of the pharmaceutical compositions of the invention. Suitable formulations may include those 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 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 present invention is administered, for example, by intravenous injection. When the TCR material of the present invention is host cells expressing a TCR of the present invention, pharmaceutically acceptable carriers for the cells for injection can include any isotonic carrier, such as normal saline (about 0.90% w / v NaCl in water, about 300 mOsm / L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA-LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or lactated Ringer's solution. In embodiments, the pharmaceutically acceptable carrier is supplemented with human serum albumen.

[0073] For purposes of the present invention, the amount or dose (e.g., number of cells if the TCR material of the present invention is one or more cells) of the TCR material of the present invention administered should be sufficient to produce 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 present invention should be sufficient to bind to 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 present invention and the condition of the animal (e.g., human), as well as the body weight of the animal (e.g., human) being treated.

[0074] Many assays for determining the dose to be administered are known in the art. For purposes of the present invention, a starting dose to be administered to a mammal can be determined using an assay that involves comparing the extent to which target cells are lysed or IFN-γ is secreted by T cells expressing a given dose of a TCR, polypeptide, or protein of the present invention when administered to the mammal, between a set of mammals that have each received different doses of T cells. The 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 various factors, e.g., 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 infusion may be, for example, about 1 x 10 6 ~Approx. 1×10 12 In certain embodiments, the number of cells may vary from 1 x 10 to 1 x 10. 6 Subcellular amounts may also be administered.

[0076] Those skilled in the art will readily appreciate that the TCR materials of the present invention may be modified in any number of ways, thereby increasing the therapeutic or prophylactic efficacy of the TCR materials of the present invention through such modifications. For example, the TCR materials of the present invention may be conjugated to a chemotherapeutic agent, either directly or indirectly via cross-linking. The practice of conjugating compounds to chemotherapeutic agents is known in the art. Those skilled in the art will recognize that sites on the TCR materials of the present invention that are not essential to the function of the TCR materials of the present invention are ideal sites for conjugating cross-linking and / or chemotherapeutic agents, as long as the cross-linking and / or chemotherapeutic agent, when bound to the TCR materials of the present invention, does not interfere with the function of the TCR materials of the present invention, i.e., their ability to bind mutant p53 or detect, treat, or prevent cancer.

[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 by theory, it is believed that the TCRs of the invention specifically bind to mutant p53, such that, when expressed by a cell, the TCR (or related polypeptides or proteins of the invention) can mediate an immune response against target cells expressing mutant 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 present invention provide any of the pharmaceutical compositions, TCRs, polypeptides, or proteins described herein, any nucleic acid or recombinant expression vector comprising a nucleotide sequence encoding any of the TCRs, polypeptides, or proteins described herein, or any host cell or population of cells comprising a recombinant vector encoding any of the TCRs, polypeptides, or proteins described herein, for use in the treatment or prevention of cancer in a mammal.

[0079] The terms "treat" and "prevent," as well as derivatives thereof, as used herein, do not necessarily mean 100%, i.e., complete treatment or prevention. Rather, there are various degrees of treatment or prevention that one of skill in the art will recognize as having potential benefits or therapeutic effects. In this regard, the methods of the present invention can provide any amount or level of cancer treatment or prevention in a mammal. Furthermore, the treatment or prevention provided by the methods of the present invention can include treatment or prevention of one or more conditions or symptoms of the cancer being treated or prevented. For example, treatment or prevention can include promoting tumor regression. Also, for purposes herein, "prevention" can include delaying the onset of cancer or a symptom or condition thereof. Alternatively, or in addition, "prevention" can include preventing or delaying the recurrence of cancer or a symptom or condition thereof.

[0080] Also provided by embodiments of the invention are methods for detecting the presence of cancer in a mammal, the methods 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 methods of the present invention for detecting cancer in a mammal, the cellular sample may be a sample comprising whole cells, a lysate thereof, or a fraction of a whole cell lysate, such as a nuclear or cytoplasmic fraction, a total protein fraction, or a nucleic acid fraction.

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

[0084] For purposes of the methods of the present invention in which a host cell or population of cells is administered, the cells may be allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal.

[0085] In the context of the methods of the present invention, cancer is selected from the group consisting 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 The cancer may be any cancer, including ovarian cancer, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. In a preferred embodiment, the cancer is a cancer that expresses mutant p53. The cancer may express p53 with 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 methods of the present invention may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, such as mice and hamsters, and lagomorph mammals, such as rabbits. Preferably, the mammal is a mammal of the order Carnivora, which includes cats and dogs. More preferably, the mammal is a mammal of the order Artiodactyla, which includes cattle and pigs, or Perissodactyla, which includes horses. Most preferably, the mammal is a mammal of the order Primates, Ceboids or Simoids (monkeys), or Anthropoids (humans and apes). A particularly preferred mammal is a human. [Example]

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

[0088] The amino acid sequences listed in Tables 1 to 3 were used in the experiments described in the following examples. In Tables 1 and 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 demonstrates the isolation and specific reactivity of four anti-mutant p53 TCRs from patient 4127.

[0094] Experiments were performed on patient 4127 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 U.S. Patent Application Publication No. 2017 / 0224800 (the "Tran method"). The method for isolating individual TCRs is described below.

[0095] Autologous PBLs were transduced with one of the TCRs shown in Figure 1 and tested after 2 weeks. Autologous DC cells were transduced at 3 x 10 4 Cells were plated at 3 × 10 cells / well 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. The 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 Figure 2.

[0096] Autologous PBLs were transduced with one of the TCRs shown in Figure 3 and treated with either DMSO (peptide vehicle) or the 15-mer 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 in wells of a flat-bottom 96-well plate. After 20 hours, the cells were co-transfected with individual HLA alleles. After 20 hours, the 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 hours) 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 gates: 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 patients in the NCI HLA database and in 5 of 9 (56%) endometrial and ovarian cancer patients in the NCI-SB (National Cancer Institute Surgery Branch). * The frequency of the 02 allele is very high according to the allelefrequencies.net website. For example, this website lists the DRB3 * The frequency of the 02 allele is Middle Eastern or North Coast of Africa It has been reported to be 0.3447 of the population.

[0101] TILs from patient 4127 were either (1) electroporated with a tandem minigene (TMG) composed of irrelevant, WT p53, or mutant p53 sequences, or (2) pulsed with a peptide vehicle (DMSO) or a purified (>95% by high-performance liquid chromatography (HPLC)) 25-amino acid peptide 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). The incubation was carried out overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. The results are shown in Figure 6.

[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 WT p53-G245 or mutant p53-G245S. 4127-O37-TCR-transduced T cells from patient 4127 were cocultured with peptide-pulsed APCs overnight at 37°C. After gating on lymphocytes, single cells, live cells, and 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 (2 x 10) to the co-culture with Cos7 cells. 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+. The 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) comprises the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO: 33) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full-length beta chain (SEQ ID NO: 36) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length alpha chain (SEQ ID NO: 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 for TCR identification: 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 (see 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) comprises the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO:43) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full-length beta chain (SEQ ID NO:46) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length alpha chain (SEQ ID NO: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 for TCR identification: 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% ​​(2 observations 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) comprises the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO: 53) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full-length beta chain (SEQ ID NO: 56) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length alpha chain (SEQ ID NO: 55) contains the sequence beginning immediately after the linker and ending at the carboxyl terminus.

[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 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). The 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. A 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 demonstrates the isolation and specific reactivity of three anti-mutant 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 U.S. 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 minimal epitopes: 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 vs. 4196-Rx1 TILs: On day 4, autologous DCs were pulsed with either the candidate minimal epitope (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 Figure 8B.

[0128] A * TP53-reactive cells can be isolated from TIL infusion bags using 02:01-restrictive TP53 tetramers: Isolation of TP53-reactive cells using 4-1BB+ sorting after co-culture with TP53 peptide was technically limited. * 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 for the TP53 TCR were identified, 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-restricted TP53 receptor identified.

[0134] T2 cells were thawed, allowed to stand for 24 hours, and then pulsed with decreasing concentrations of HPLC minimal epitopes for 2 hours. The 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 collected and plated (1 × 10 5The R175H minimal epitope was pulsed onto target cells (HPLC, 5 μg / mL) for 2 hours. The cells were washed twice and 2 × 10 4 The target cells were co-cultured with TP53 TCR-transduced cells for 20 hours (Patient 4196). IFN-γ ELISPOT was used to read the response. The results are shown in Figure 11. Further experimental data regarding HLA-A2 and TP53 expression on target cells and the reactivity of the TCRs 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 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: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 immediately 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) comprises the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO: 83) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full-length beta chain (SEQ ID NO: 86) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length alpha chain (SEQ ID NO: 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) comprises the sequence beginning at the amino terminus and ending just before the start of the beta chain constant region. The alpha chain variable region (SEQ ID NO: 93) contains the sequence beginning immediately after the linker and ending just before the start of the alpha chain constant region. The full-length beta chain (SEQ ID NO: 96) contains the sequence beginning at the amino terminus and ending just before the start of the linker. The full-length alpha chain (SEQ ID NO: 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 demonstrates the isolation of 11 anti-mutated p53 TCRs from patient 4238.

[0147] For patient 4238, the experiment 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 consisting of irrelevant or mutant p53 sequences. Co-culture was performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. The 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 Figure 14.

[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 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F10 with R248Q long peptide and sorting of 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F10 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F10 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 58.3% (7 out of 12 pairs observed) 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F10 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 58.3% (7 out of 12 pairs observed) 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F11 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F11 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F11 with R248Q long peptide and sorting of 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

[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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F11 with R248Q long peptide and sorting of 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F17 with R248Q long peptide and sorting of 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F17 with R248Q long peptide and sorting of 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

[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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4238-F17 with R248Q long peptide and sorting of 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

[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 demonstrates the isolation of two anti-mutant p53 TCRs from patient 4253.

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

[0208] TIL fragments (F1–F24; n = 24) from patient 4253 were cocultured with autologous APCs that were (1) pulsed with peptide vehicle (DMSO), (2) pulsed with a 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. Cocultures were performed overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. The results are shown in Figure 16. F15 was selected 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. The methods used to isolate TCRs are described below. TCR name: 4253-TIL-TCR1 Recognizing p53 mutation: R248W Method: A simplified universal screening method for p53 "hot spot" mutations (evaluating only mutant TMG and R248W long peptides) Co-culture for TCR identification: Co-culture of 4253-F15 with p53-R248W long peptide and sorting of CD3+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 5.6% (2 observations 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. The methods used to isolate TCRs are described below. TCR name: 4253-TIL-TCR2 Recognizing p53 mutation: R248W Method: A simplified universal screening method for p53 "hot spot" mutations (evaluating only mutant TMG and R248W long peptides) Co-culture for TCR identification: Co-culture of 4253-F15 with p53-R248W long peptide and sorting of CD3+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 91.7% (observed 33 times 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 demonstrates the identification of anti-mutant p53 T cells in patient 4273 by co-culturing autologous APCs induced to express mutant p53 within autologous T cells ("p53 hotspot mutation universal screen"). This example also demonstrates the isolation of two anti-mutant p53 TCRs from patient 4273.

[0220] For patient 4273, experiments were 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 Figure 18.

[0222] TIL fragments (F1–F24, n = 24) from patient 4273 were cocultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25-amino acid peptides composed of the WT p53-R248 or mutant p53-R248W sequences. Cocultures 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 F15-induced responses to LP and TMG were comparable and primarily driven by CD4 T cells. Therefore, 4273-F15 was cocultured with APCs pulsed with R248W LP. The following day, CD4+41BB+ T cells were single-cell sorted into wells of a 96-well PCR plate at 1 cell per well. Each well of the PCR plate contained an RT-PCR solution, which amplifies the alpha and beta CDR3 regions of the TCR in the same solution. The wells of the plate were then split into two 96-well PCR plates, and a second PCR round was performed to amplify either CDR3 alpha or CDR3 beta in separate reactions. The PCR products from each well (a total of 192 PCR products mapped to each well—alpha or beta) were sequenced by Sanger sequencing. The nucleotide sequences were 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 was determined and fused to the CDR3 and junctions (J or DJ) from the translated sequence. The variable sequences were fused to mouse constant sequences, and the rearranged TCR alpha and TCR beta were 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 unrelated mutation, the WT p53 sequence, or a mutant p53 sequence containing p53-R248W. Medium alone and PMA and ionomycin served as negative and positive controls, respectively. TILs from patient 4273 (fragment cultures 8 and 15) were cocultured with TMG-transfected APCs overnight at 37°C. 4-1BB expression was assessed by flow cytometry after gating on lymphocytes, viable cells (PI negative), and CD3+ (T cells). The 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 cocultured 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, viable cells (PI negative), and CD3+ (T cells). The 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 by 14 amino acids. TILs (fragment culture 15) from patient 4273 with specificity for p53-R248W were cocultured 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 peptide (wt p53-R248 and mutant p53-R248W) served as additional controls for the 15-amino acid peptide. 4-1BB expression was assessed by flow cytometry after gating lymphocytes → live cells (PI negative) → CD3+ (T cells) → CD4+CD8-. The results are shown in Figure 58.

[0227] Cos7 cells (2.5×10 4Cells (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 4273 and either wild-type or mutant TP53 TMG, with or without the p53-R248W neoantigen. The following day, TILs (fragment culture 15) with specificity for p53-R248W from patient 4273 were co-cultured with transfected Cos7 cells overnight at 37°C. IFN-γ secretion was assessed by ELISA. The results are shown in Figure 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 the wild-type p53-R248 sequence or the mutant p53-R248W sequence. Medium alone, PMA, and ionomycin served as negative and positive controls, respectively. Co-cultures were performed overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. Results are shown in Figure 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) comprises the sequence beginning at the amino terminus and ending just before the start 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 the 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 the sequence and (2) did not have a stop codon in the sequence), with a pairing frequency of 80.2%. Of these pairs, 43 had the CDR3A / CDR3B combination to create 4273-TP53-R248W-TCR1a1 (55.8% of productive pairs). Of these pairs, 30 had the CDR3A / CDR3B combination to create 4273-TP53-R248W-TCR1a2 (39% of productive pairs). In summary, CDR3A and CDR3B of 4273-TP53-R248W-TCR1a1 were observed 50 and 83 times, respectively, out of 96 wells. In summary, CDR3A and CDR3B of 4273-TP53-R248W-TCR1a2 were observed 33 and 83 times, respectively, out of 96 wells.

[0234] [Table 19]

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

[0236] For patient 4149, experiments were performed as described in Figures 21-24. The TCR was discovered using the Tran method. 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 PBLs from patient 4149 and cocultured with autologous APCs (1) electroporated with TMG composed of irrelevant, wild-type p53, or mutant p53 sequences, or (2) pulsed with peptide vehicle (DMSO) or purified (>95% purified by HPLC) 25-amino acid peptides composed of the wild-type p53-Y220 or mutant p53-Y220C sequences. A combination of PMA and ionomycin served as a positive control. Cocultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. The 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). The results are shown in Figure 22.

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

[0239] [Table E]

[0240] Putative p53 recognized by 4149-F11 Y220C Minimal epitope mapping: Autologous DCs were pulsed with the peptide (10 μg / mL) and then incubated with granulocyte-macrophage colonies. TILs were incubated overnight in stimulatory factors (GM-CSF) and IL-4. TILs were incubated in 500 CU / mL IL-2 for 2-3 days. 2 × 10 4 TIL and 10 5 Target cells were co-cultured overnight at 37°C. IFN-γ was measured by ELISPOT. The results are shown in Table F. 4-1BB expression was measured by FACS gated on lymphocytes \PI(negative)CD3+\CD3+CD4+. The 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, the 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 in one set of wells. Cells not transfected with TMG were pulsed with 10 μg / mL of p53-Y220C 15-mer peptide at 37°C for 2–3 hours. After washing, 4149-TCRa2b2-transfected T cells (10 cells / well) on day 14 of the second REP were transfected with 4149-TCRa2b2. 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 Figure 24. Predictions by NetMHCIIpan: cbs.dtu.dk / services / NetMHCIIpan / (Table G).

[0243] [Table G]

[0244] In the NCI HLA database, 1367 (37%) of 3719 DRB patients and in the NCI-SB, 5 (56%) of 9 endometrial and ovarian cancer patients 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). The first 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 starting at the amino terminus and ending just before the start of the alpha chain constant region. The beta chain variable region (SEQ ID NO: 64) contains the sequence starting immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO: 65) contains the sequence starting at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO: 66) contains the sequence starting just after the linker and ending 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 universal screening of p53 "hotspot" mutations Co-culture to identify TCR: Co-culture of 4149-F11 TIL fragment with p53-Y220C long peptide and sorting of CD4+41BB+ T cells. Method for identifying TCRs: Pairing frequency. CD4+41BB+ sorted T cells were expanded using REP (Rapid Expansion Protocol), and the resulting T cell cultures were subjected to TCRAD (alpha) and TCRB (beta) deep sequencing by Adaptive Biotechnologies. In a matrix of four TCRs in total, the top two TCR alphas paired with the top two TCR betas. The second TCR alpha and the second TCR beta were reactive TCRs and were therefore designated TCRa2b2. Abundance of TCR among all TCRs: as follows 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 demonstrates the identification of anti-mutant p53 T cells in patient 4213 by co-culturing autologous APCs induced to express mutant p53 within autologous T cells ("p53 hotspot mutation universal screen"). This example also demonstrates the isolation of 12 anti-mutant 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 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. After gating on lymphocytes → live cells (PI negative) → CD3+ (T cells), 4-1BB expression was assessed by flow cytometry. 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. After gating on lymphocytes → live cells (PI negative) → CD3+ (T cells), 4-1BB expression was assessed by flow cytometry. 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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 for TCR identification: Co-culture of 4213-F2 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 8.3% (2 observed in 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4213-F2 with R248Q long peptide and sorting of 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4213-F2 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 70.8% (17 observations 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4213-F24 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods 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 in 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4213-F24 with R248Q long peptide and sorting of CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 84.1% (37 observations out of 44 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0276] [ka]

[0277] Statistics for TCR4213-F24-TCRa2 in 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 9.5% (6 observed 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 3.2% (2 observations out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0296] [ka]

[0297] Statistics for TCR4213-PBL-TCR4a1 of 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 3.2% (2 observations out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0301] [ka]

[0302] Statistics for TCR4213-PBL-TCR4a2 in patient 4213 are listed 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods 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 in 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 3.2% (2 observations out of 63 pairs) TCR orientation: alpha-beta Expression vector: SB transposon

[0311] [ka]

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

[0313] [Table 32]

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

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

[0316] TIL fragments (F1-F24, n=24) from patient 4268 were cocultured with autologous APCs electroporated with TMG composed of irrelevant, WT p53, or mutant p53 sequences. Cocultures 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 the 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 Figure 28.

[0318] TIL fragments (F1–F24, n = 24) from patient 4268 were incubated with peptide vehicle (D The cells were co-cultured with autologous APCs pulsed with purified (>95% by HPLC) 25-amino acid peptides composed of either the wild-type p53-R248 or mutant p53-R248Q sequences. Co-culture was performed overnight at 37°C. After gating on lymphocytes, viable cells (PI negative), and CD3+ (T cells), 4-1BB expression was assessed by flow cytometry. The results are shown in Figure 29. The source of TCRs after sorting CD4+41BB+ T cells was the TIL fragments F18 and F19.

[0319] TIL fragments (F1–F24, n = 24) from patient 4268 were cocultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25-amino acid peptides composed of the WT p53-R248 or mutant p53-R248Q sequences. Cocultures were performed overnight at 37°C. After gating on lymphocytes, viable cells (PI negative), and CD3+ (T cells), 4-1BB expression was assessed by flow cytometry. 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 of 4268-F7 and 4268-F8 with p53-R248Q long peptide and sorting of 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 of 4268-F7 and 4268-F8 with p53-R248Q long peptide and sorting of CD8+41BB+ T cells. Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4268-F15 with p53-R248Q long peptide and sorting of CD8+41BB+ T cells Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 88.6% (31 observations 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4268-F18 with p53-R248Q long peptide and sorting of CD4+41BB+ T cells Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: Co-culture of 4268-F19 with p53-mut-TMG and sorting of CD4+41BB+ T cells Methods 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 demonstrates the identification of anti-mutant p53 T cells in patient 4266 by co-culturing autologous APCs induced to express mutant p53 within autologous T cells ("p53 hotspot mutation universal screen"). This example also demonstrates the isolation of four anti-mutant p53 TCRs from patient 4266.

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

[0347] TIL fragments (F1–F24, n = 24) from patient 4266 were cocultured with autologous APCs electroporated with TMG composed of irrelevant, WT p53, or mutant p53 sequences. Cocultures 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 APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25-amino acid peptides composed of the 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 in wells of a flat-bottom 96-well plate (cells / well). The following day, individual HLA alleles from patient 4266 were co-transfected into the cells. The following day, cells were pulsed with no peptide, DMSO, wild-type p53-R248 peptide SSCMGGMNRR (SEQ ID NO: 590), or mutant p53-R248W peptide SSCMGGMNWR (SEQ ID NO: 591) at 1 μg / mL for 2 hours at 37°C. TIL cultures from subject 4266 (10 5 (individually, 1000 cells / well) were added to the wells and co-cultured overnight at 37°C. After gating 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, which have putative specificity for p53-R248W identified from 4266-TILs, were cocultured with autologous APCs pulsed with peptide vehicle (DMSO) or a purified (>95% purified by HPLC) 25-amino acid peptide composed of the WT p53-R248 sequence or the mutant p53-R248W sequence. Medium alone, PMA, and ionomycin served as negative and positive controls, respectively. Cocultures 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+. The results are shown in Figure 54.

[0351] A tumor cell (TC) line was established from a xenograft tumor fragment excised from patient 4266 and then serially passaged in immunocompromised mice (TC#4266). T cells (10 ) expressing mock (no TCR) or p53-R248W-specific TCRs (4266-TCR2, 4266-TCR3, or 4266-TCR4) were cultured in 10 . 5 TC#4266 cells were co-cultured overnight at 37°C. 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-culture at a final concentration of 5 μg / mL. Peptides were incubated at 1 μg / mL, and excess peptide was washed away after incubation. Medium alone (no TC) and PMA and ionomycin were used as negative and positive controls, respectively. 4-1BB expression was assessed 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: 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 sorted CD8+41BB+ T cells. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 17.0% (9 observations 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: 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 sorted 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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 for TCR identification: 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 sorted CD8+41BB+ T cells. Methods 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) comprises the sequence beginning at the amino terminus and ending just before the start 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] The screening methods described below were used to identify cancer-reactive T cells. 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. Methods 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 "hotspot" mutations.

[0373] A summary of T cell response to p53 "hot spot" mutations 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 the treatment of a patient with p53 mutation-responsive TILs.

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

[0377] [Table 43]

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

[0379] Autologous APCs were transfected with TMG encoding an unrelated mutation, the wild-type p53 sequence, or a mutant p53 sequence containing R175H. Medium alone and PMA and ionomycin served as negative and positive controls, respectively. The following day, TILs from patient 4141 (fragment culture 12) were cocultured with TMG-transfected APCs overnight at 37°C. IFN-γ secretion was assessed by ELISPOT. 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 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 (fragment culture 12) with specificity for p53-R175H from patient 4141 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 Figure 39.

[0381] Mock (no TCR) or 4141-TCR1a2-expressing T cells were injected into 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 hours at 37°C. Medium alone, PMA, and ionomycin were negative and positive controls, respectively. Co-culture was performed overnight at 37°C. IFN-γ secretion was assessed by ELISA. The results are shown in Figure 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 unmanipulated or overexpressed full-length p53-R175H protein. To capture cytokines within T cells, inhibitors of secretion (monensin and brefeldin A) were added to the coculture. After 6 hours of coculture, 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 cocultures 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 start 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 start 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: 4141 infusion bag TILs were co-cultured with p53mutTMG and CD8+41BB+ T cells were sorted. 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 listed in Table 44 below.

[0387] [Table 44]

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

[0389] TIL fragment cultures (#6) from patient 4259 were cocultured with autologous APCs (1) electroporated with TMG composed of irrelevant, wild-type p53, or mutant p53 sequences, or (2) pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25-amino acid peptides composed of the wild-type p53-Y220 or mutant p53-Y220C sequences. Cocultures 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 WT p53-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 cocultured 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). The results are shown in Figure 44.

[0392] Cos7 cells (2.5×10 4 Cells (cells / well) were plated into wells of a flat-bottom 96-well plate. 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 the transfected Cos7 cells for 2 hours. Excess peptide was washed away. TIL fragment culture number 6 from patient 4259 was added (1 0 5 Cells / well). Co-cultures were incubated overnight at 37°C. 4-1BB expression was assayed by flow cytometry after gating on lymphocytes → live cells → CD3+ (T cells) → CD8-CD4+. The results are shown in Figure 45.

[0393] A 25-amino acid peptide corresponding to the sequence of WT p53-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, viable cells (PI negative), and CD3+ (T cells), 4-1BB expression was assessed by flow cytometry. The transduced TCR was measured using 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 transfected 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 TC#4259 cells overnight at 37°C. TC#4259 cells were incubated 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) for 2 hours at 37°C. Antibodies were maintained in the co-culture at a final concentration of 5 μg / mL. Peptides were incubated at 10 μg / mL, and excess peptide was washed away 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). The 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) comprises the sequence beginning at the amino terminus and ending just before the start 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 Recognizing 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% (36 observations 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 demonstrates the isolation and specific reactivity of a TCR from patient 4285.

[0402] TIL fragments (F1-F22 and F24, n=23) from patient 4285 were cocultured with autologous APCs electroporated with TMG composed of irrelevant, WT p53, or mutant p53 sequences. Cocultures 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 cocultured with autologous APCs pulsed with peptide vehicle (DMSO) or purified (>95% by HPLC) 25-amino acid peptides composed of the WT p53-R175 sequence or the mutant p53-R175H sequence. Cocultures were performed overnight at 37°C. IFN-γ secretion was assessed using an ELISPOT assay. Results are shown in Figure 48. 4-1BB expression was assessed by flow cytometry after gating on 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 TIL fragment F6.

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

[0405] [Table 47]

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

[0407] Autologous APCs were pulsed with decreasing concentrations of 25- or 15-amino acid peptides corresponding to the sequences of wild-type or mutant p53-R175H for 2 hours at 37°C. 4285-TCR1-transfected T cells from patient 4285 were cocultured with peptide-pulsed APCs overnight at 37°C. After gating on lymphocytes → live cells → CD3+ (T cells) → CD8-CD4+, 4-1BB expression was assayed by flow cytometry. The results are shown in Figure 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 the sequence starting at the amino terminus and ending just before the start of the alpha chain constant region. The beta chain variable region (SEQ ID NO:494) contains the sequence starting immediately after the linker and ending just before the start of the beta chain constant region. The full-length alpha chain (SEQ ID NO:495) contains the sequence starting at the amino terminus and ending just before the start of the linker. The full-length beta chain (SEQ ID NO:496) contains the 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. Methods for identifying TCR: Single-cell RT-PCR TCR abundance among all paired TCRs: 81.8% (36 observations 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 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," "an," "the," and "at least one," and similar referents, unless otherwise specified herein or clearly contradicted by context, should be construed to cover both the singular and the plural. The use of "at least one" (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 herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are used in conjunction with "including," "having," "including," and "containing" to refer to any combination of the listed items, particularly those that are not part of the "list" or "item." Unless otherwise expressly stated, terms should be construed as open-ended (i.e., meaning "including, but not limited to"). The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method for individually referencing each separate value within the range, unless otherwise specified herein, and each separate value is incorporated into the specification as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to further elucidate the invention and does not pose a limitation on the scope of the invention, unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0424] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be employed by those skilled in the art, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

Claims

1. An isolated or purified T cell receptor (TCR) having antigen specificity for human p53 having a G245S mutation, wherein the TCR comprises: (1) the amino acid sequence of an α chain complementarity-determining region (CDR) 1 of SEQ ID NO: 27, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 28, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 29, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 30, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 31, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 32; (2) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 37, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 38, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 39, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 40, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 41, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 42; (3) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 47, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 48, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 49, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 50, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 51, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 52; or (4) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 457, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 458, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 459, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 460, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 461, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 462; Including, TCR. (1) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 33, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 34; (2) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44; (3) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 53 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 54; (4) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 463, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 464; (5) an amino acid sequence that is at least 95% identical to amino acids 21 to 130 of SEQ ID NO: 33 and at least 95% identical to amino acids 22 to 131 of SEQ ID NO: 34; (6) an amino acid sequence that is at least 95% identical to amino acids 22 to 135 of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to amino acids 20 to 132 of SEQ ID NO: 44; (7) an amino acid sequence that is at least 95% identical to amino acids 19 to 127 of SEQ ID NO: 53 and at least 95% identical to amino acids 24 to 134 of SEQ ID NO: 54; or (8) an amino acid sequence that is at least 95% identical to amino acids 21-131 of SEQ ID NO: 463 and at least 95% identical to amino acids 22-140 of SEQ ID NO: 464; 2. The TCR of claim 1, comprising:

3. (1) Both of SEQ ID NOs: 33 and 34; (2) both of SEQ ID NOs: 43-44; (3) both of SEQ ID NOs: 53-54; (4) Both of SEQ ID NOs: 463-464; (5) both amino acids 21 to 130 of SEQ ID NO: 33 and amino acids 22 to 131 of SEQ ID NO: 34; (6) both amino acids 22 to 135 of SEQ ID NO: 43 and amino acids 20 to 132 of SEQ ID NO: 44; (7) both amino acids 19 to 127 of SEQ ID NO: 53 and amino acids 24 to 134 of SEQ ID NO: 54; or (8) both amino acids 21-131 of SEQ ID NO: 463 and amino acids 22-140 of SEQ ID NO: 464; The TCR of claim 1 or 2, comprising the amino acid sequence:

4. (1) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 35 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 36; (2) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 45 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 46; (3) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 55 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 56; (4) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 465, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 466; (5) an amino acid sequence that is at least 95% identical to amino acids 21 to 267 of SEQ ID NO: 35 and at least 95% identical to amino acids 22 to 304 of SEQ ID NO: 36; (6) an amino acid sequence that is at least 95% identical to amino acids 22 to 272 of SEQ ID NO: 45 and an amino acid sequence that is at least 95% identical to amino acids 22 to 305 of SEQ ID NO: 46; (7) an amino acid sequence that is at least 95% identical to amino acids 19 to 264 of SEQ ID NO: 55 and at least 95% identical to amino acids 24 to 307 of SEQ ID NO: 56; or (8) an amino acid sequence that is at least 95% identical to amino acids 21-268 of SEQ ID NO: 465 and at least 95% identical to amino acids 22-313 of SEQ ID NO: 466; The TCR of any one of claims 1 to 3, comprising:

5. (1) both of SEQ ID NOs: 35 and 36; (2) both of SEQ ID NOs: 45 and 46; (3) both of SEQ ID NOs: 55 and 56; (4) Both of SEQ ID NOs: 465-466; (5) both amino acids 21 to 267 of SEQ ID NO: 35 and amino acids 22 to 304 of SEQ ID NO: 36; (6) both amino acids 22-272 of SEQ ID NO: 45 and amino acids 22-305 of SEQ ID NO: 46; (7) both amino acids 19 to 264 of SEQ ID NO: 55 and amino acids 24 to 307 of SEQ ID NO: 56; or (8) both amino acids 21 to 268 of SEQ ID NO: 465 and amino acids 22 to 313 of SEQ ID NO: 466; The TCR according to any one of claims 1 to 3, comprising the amino acid sequence:

6. 10. An isolated or purified polypeptide comprising a functional portion of a TCR according to any one of claims 1 to 5, wherein said functional portion comprises: (1) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 27, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 28, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 29, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 30, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 31, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 32; (2) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 37, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 38, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 39, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 40, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 41, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 42; (3) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 47, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 48, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 49, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 50, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 51, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 52; or (4) the amino acid sequence of an α chain CDR1 of SEQ ID NO: 457, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 458, the amino acid sequence of an α chain CDR3 of SEQ ID NO: 459, the amino acid sequence of a β chain CDR1 of SEQ ID NO: 460, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 461, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 462; A polypeptide comprising: (1) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 33, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 34; (2) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 44; (3) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 53 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 54; (4) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 463, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 464; (5) an amino acid sequence that is at least 95% identical to amino acids 21 to 130 of SEQ ID NO: 33 and at least 95% identical to amino acids 22 to 131 of SEQ ID NO: 34; (6) an amino acid sequence that is at least 95% identical to amino acids 22 to 135 of SEQ ID NO: 43 and an amino acid sequence that is at least 95% identical to amino acids 20 to 132 of SEQ ID NO: 44; (7) an amino acid sequence that is at least 95% identical to amino acids 19 to 127 of SEQ ID NO: 53 and at least 95% identical to amino acids 24 to 134 of SEQ ID NO: 54; or (8) an amino acid sequence that is at least 95% identical to amino acids 21-131 of SEQ ID NO: 463 and at least 95% identical to amino acids 22-140 of SEQ ID NO: 464; The polypeptide of claim 6, comprising:

8. (1) both of SEQ ID NOs: 33 and 34; (2) both of SEQ ID NOs: 43-44; (3) both of SEQ ID NOs: 53-54; (4) Both of SEQ ID NOs: 463-464; (5) both amino acids 21 to 130 of SEQ ID NO: 33 and amino acids 22 to 131 of SEQ ID NO: 34; (6) both amino acids 22 to 135 of SEQ ID NO: 43 and amino acids 20 to 132 of SEQ ID NO: 44; (7) both amino acids 19 to 127 of SEQ ID NO: 53 and amino acids 24 to 134 of SEQ ID NO: 54; or (8) both amino acids 21-131 of SEQ ID NO: 463 and amino acids 22-140 of SEQ ID NO: 464; 7. The polypeptide of claim 6, comprising the amino acid sequence: (1) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:36; (2) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 45 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 46; (3) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 55 and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 56; (4) an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 465, and an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 466; (5) an amino acid sequence that is at least 95% identical to amino acids 21 to 267 of SEQ ID NO: 35 and at least 95% identical to amino acids 22 to 304 of SEQ ID NO: 36; (6) an amino acid sequence that is at least 95% identical to amino acids 22 to 272 of SEQ ID NO: 45 and an amino acid sequence that is at least 95% identical to amino acids 22 to 305 of SEQ ID NO: 46; (7) an amino acid sequence that is at least 95% identical to amino acids 19 to 264 of SEQ ID NO: 55 and at least 95% identical to amino acids 24 to 307 of SEQ ID NO: 56; or (8) an amino acid sequence that is at least 95% identical to amino acids 21-268 of SEQ ID NO: 465 and at least 95% identical to amino acids 22-313 of SEQ ID NO: 466; The polypeptide according to any one of claims 6 to 8, comprising:

10. (1) both of SEQ ID NOs: 35 and 36; (2) both of SEQ ID NOs: 45 and 46; (3) both of SEQ ID NOs: 55 and 56; (4) Both of SEQ ID NOs: 465-466; (5) both amino acids 21 to 267 of SEQ ID NO: 35 and amino acids 22 to 304 of SEQ ID NO: 36; (6) both amino acids 22-272 of SEQ ID NO: 45 and amino acids 22-305 of SEQ ID NO: 46; (7) both amino acids 19 to 264 of SEQ ID NO: 55 and amino acids 24 to 307 of SEQ ID NO: 56; or (8) both amino acids 21 to 268 of SEQ ID NO: 465 and amino acids 22 to 313 of SEQ ID NO: 466; The polypeptide according to any one of claims 6 to 8, comprising the amino acid sequence:

11. An isolated or purified protein comprising a functional portion of a TCR having antigen specificity for human p53 having a G245S mutation, wherein the functional portion is: (1) a first polypeptide chain comprising the amino acid sequence of an α chain CDR1 of SEQ ID NO: 27, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 28, and the amino acid sequence of an α chain CDR3 of SEQ ID NO: 29, and a second polypeptide chain comprising the amino acid sequence of a β chain CDR1 of SEQ ID NO: 30, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 31, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 32; (2) a first polypeptide chain comprising the amino acid sequence of an α chain CDR1 of SEQ ID NO: 37, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 38, and the amino acid sequence of an α chain CDR3 of SEQ ID NO: 39, and a second polypeptide chain comprising the amino acid sequence of a β chain CDR1 of SEQ ID NO: 40, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 41, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 42; (3) a first polypeptide chain comprising the amino acid sequence of an alpha chain CDR1 of SEQ ID NO: 47, the amino acid sequence of an alpha chain CDR2 of SEQ ID NO: 48, and the amino acid sequence of an alpha chain CDR3 of SEQ ID NO: 49, and a second polypeptide chain comprising the amino acid sequence of a beta chain CDR1 of SEQ ID NO: 50, the amino acid sequence of a beta chain CDR2 of SEQ ID NO: 51, and the amino acid sequence of a beta chain CDR3 of SEQ ID NO: 52; or (4) A first polypeptide chain comprising the amino acid sequence of an α chain CDR1 of SEQ ID NO: 457, the amino acid sequence of an α chain CDR2 of SEQ ID NO: 458, and the amino acid sequence of an α chain CDR3 of SEQ ID NO: 459, and a second polypeptide chain comprising the amino acid sequence of a β chain CDR1 of SEQ ID NO: 460, the amino acid sequence of a β chain CDR2 of SEQ ID NO: 461, and the amino acid sequence of a β chain CDR3 of SEQ ID NO: 462; Proteins, including:

12. The protein of claim 11, (1) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:33, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:34; (2) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:43, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:44; (3) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:53, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:54; (4) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 463, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 464; (5) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 21-130 of SEQ ID NO:33, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-131 of SEQ ID NO:34; (6) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-135 of SEQ ID NO:43, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 20-132 of SEQ ID NO:44; (7) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 19-127 of SEQ ID NO:53, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 24-134 of SEQ ID NO:54; or (8) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 21-131 of SEQ ID NO:463, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-140 of SEQ ID NO:464; protein.

13. The protein of claim 11, (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 33 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 34; (2) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:43 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:44; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:53 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:54; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 463 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 464; (5) the first polypeptide chain comprises amino acids 21-130 of SEQ ID NO: 33, and the second polypeptide chain comprises amino acids 22-131 of SEQ ID NO: 34; (6) the first polypeptide chain comprises amino acids 22-135 of SEQ ID NO:43, and the second polypeptide chain comprises amino acids 20-132 of SEQ ID NO:44; (7) the first polypeptide chain comprises amino acids 19-127 of SEQ ID NO: 53 and the second polypeptide chain comprises amino acids 24-134 of SEQ ID NO: 54; or (8) A protein wherein the first polypeptide chain comprises amino acids 21-131 of SEQ ID NO: 463 and the second polypeptide chain comprises amino acids 22-140 of SEQ ID NO:

464.

14. The protein according to any one of claims 11 to 13, (1) the first polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:35, and the second polypeptide chain comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:36; (2) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:45, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:46; (3) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:55, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:56; (4) the first polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:465, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:466; (5) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 21-267 of SEQ ID NO:35, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-304 of SEQ ID NO:36; (6) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-272 of SEQ ID NO:45, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-305 of SEQ ID NO:46; (7) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 19-264 of SEQ ID NO:55, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-307 of SEQ ID NO:56; or (8) the first polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 21-268 of SEQ ID NO:465, and the second polypeptide chain comprises an amino acid sequence at least 95% identical to amino acids 22-313 of SEQ ID NO:466; protein.

15. The protein according to any one of claims 11 to 13, (1) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 35 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 36; (2) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:45 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:46; (3) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:55 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:56; (4) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 465 and the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 466; (5) the first polypeptide chain comprises amino acids 21-267 of SEQ ID NO: 35, and the second polypeptide chain comprises amino acids 22-304 of SEQ ID NO: 36; (6) the first polypeptide chain comprises amino acids 22-272 of SEQ ID NO:45, and the second polypeptide chain comprises amino acids 22-305 of SEQ ID NO:46; (7) the first polypeptide chain comprises amino acids 19-264 of SEQ ID NO: 55, and the second polypeptide chain comprises amino acids 22-307 of SEQ ID NO: 56; or (8) A protein wherein the first polypeptide chain comprises amino acids 21-268 of SEQ ID NO: 465 and the second polypeptide chain comprises amino acids 22-313 of SEQ ID NO:

466.

16. An isolated or purified nucleic acid comprising a nucleotide sequence encoding a TCR according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 10, or a protein according to any one of claims 11 to 15.

17. A recombinant expression vector comprising the nucleic acid of claim 16.

18. 18. An isolated or purified host cell comprising the recombinant expression vector of claim 17.

19. 20. A population of isolated or purified cells comprising the host cell of claim 18.

20. 19. A pharmaceutical composition comprising: (a) a TCR according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 10, a protein according to any one of claims 11 to 15, a nucleic acid according to claim 16, a recombinant expression vector according to claim 17, a host cell according to claim 18, or a population of cells according to claim 19; and (b) a pharmaceutically acceptable carrier.

21. 1. A method for detecting the presence of cancer in a mammal, comprising: (a) contacting a sample containing cells of the cancer with the TCR of any one of claims 1 to 5, the polypeptide of any one of claims 6 to 10, the protein of any one of claims 11 to 15, the nucleic acid of claim 16, the recombinant expression vector of claim 17, the host cell of claim 18, the population of cells of claim 19, or the pharmaceutical composition of claim 20, thereby forming a complex; (b) detecting the complex; and Including, The method, wherein detection of said complex indicates the presence of cancer in said mammal.

22. 20. A pharmaceutical composition comprising a TCR according to any one of claims 1 to 5, a polypeptide according to any one of claims 6 to 10, a protein according to any one of claims 11 to 15, a nucleic acid according to claim 16, a recombinant expression vector according to claim 17, a host cell according to claim 18, or a population of cells according to claim 19, for use in treating or preventing cancer in a mammal.

23. The pharmaceutical composition of claim 22, wherein the population of cells is autologous to the mammal.

24. The pharmaceutical composition of claim 22, wherein the population of cells is homologous to the mammal.

25. 22. The method of claim 21, wherein the cancer is an epithelial cancer.

26. 22. The method of claim 21, wherein the cancer is bile duct cancer, 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.

27. ​​A pharmaceutical composition described in any one of claims 22 to 24, wherein the cancer is epithelial cancer.

28. The pharmaceutical composition of any one of claims 22 to 24, wherein the cancer is bile duct cancer, 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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