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. 2022 Jun 23;10(8):932–946. doi: 10.1158/2326-6066.CIR-22-0040

Figure 2.

Figure 2. Characterization of mutant p53–reactive TCRs. A, Tumor-cell recognition by p53Y220C-TCR (Y220C-TCR). Y220C-TCR was expressed in healthy donor PBLs and was tested against a panel of tumor cell lines with different TP53 mutations and HLAs. Following coculture with tumor cell lines, cell surface upregulation of 4–1BB on the T cells was measured by flow cytometry. Mock transduced T cells were included as a negative control (mean ± SEM, n = 3). B, Titration curve showing the avidity of Y220C-TCR against the WT and mutant Y220C ME. 4–1BB upregulation in healthy donor PBL transduced with Y220C-TCR following coculture with A*02:01+ T2 cells pulsed with the serially diluted ME was measured by flow cytometry (n = 1). C, Comparison of 4 HLA-A*02:01-restricted TCRs targeting p53R175H based on tumor cell reactivity. 4–1BB upregulation in p53R175H-TCR+CD8+ cells was measured by flow cytometry (mean ± SEM, n = 2). D, Titration curves for 4 TCRs targeting p53R175H. HLA-A*02:01+ T2 cells were pulsed with the serially diluted p53R175H ME and cocultured with TCR-transduced T cells. IFNγ secretion was measured by ELISA (mean ± SEM, n = 3). Statistical analysis by two-way ANOVA. Preclinical ACT of NSG mice bearing TYK-nu cancer cells using the R175H-TCR (4196-AV6/BV11)-engineered human PBL. Tumor size was calculated as the product of two perpendicular measurements. Tumor measurement was discontinued when the first mouse was euthanized [mean ± SEM, n = 4 (E and F), 5 (G, H, and J), and 10 (I)]. Donor information, transduction efficiency (TX; %), and CD8+ cell frequency (%) are given. E and G, Tumor growth following ACT of two different healthy donor PBLs transduced with the R175H-TCR or the irrelevant Y220C-TCR. I and J, Tumor growth following ACT of patient 4349’s PBL (1 or 2 × 107 cells) transduced with the R175H-TCR or untransduced (2 × 107 cells). J, Mice injected with either TYK-nu cells or the control 4259 PDX cells were treated with untransduced T cells, R175H-TCR–engineered T cells, or vehicle (PBS). Statistical analyses by two-way ANOVA (D, E, G, I, J) and by log-rank tests (F and H). *, P < 0.05; **, P < 0.01; ***, P < 0.001. A–D were independently repeated at least once.

Characterization of mutant p53–reactive TCRs. A, Tumor-cell recognition by p53Y220C-TCR (Y220C-TCR). Y220C-TCR was expressed in healthy donor PBLs and was tested against a panel of tumor cell lines with different TP53 mutations and HLAs. Following coculture with tumor cell lines, cell surface upregulation of 4–1BB on the T cells was measured by flow cytometry. Mock transduced T cells were included as a negative control (mean ± SEM, n = 3). B, Titration curve showing the avidity of Y220C-TCR against the WT and mutant Y220C ME. 4–1BB upregulation in healthy donor PBL transduced with Y220C-TCR following coculture with A*02:01+ T2 cells pulsed with the serially diluted ME was measured by flow cytometry (n = 1). C, Comparison of 4 HLA-A*02:01-restricted TCRs targeting p53R175H based on tumor cell reactivity. 4–1BB upregulation in p53R175H-TCR+CD8+ cells was measured by flow cytometry (mean ± SEM, n = 2). D, Titration curves for 4 TCRs targeting p53R175H. HLA-A*02:01+ T2 cells were pulsed with the serially diluted p53R175H ME and cocultured with TCR-transduced T cells. IFNγ secretion was measured by ELISA (mean ± SEM, n = 3). Preclinical ACT of NSG mice bearing TYK-nu cancer cells using the R175H-TCR (4196-AV6/BV11)-engineered human PBL. Tumor size was calculated as the product of two perpendicular measurements. Tumor measurement was discontinued when the first mouse was euthanized [mean ± SEM, n = 4 (E and F), 5 (G, H, and J), and 10 (I)]. Donor information, transduction efficiency (TX; %), and CD8+ cell frequency (%) are given. E and G, Tumor growth following ACT of two different healthy donor PBLs transduced with the R175H-TCR or the irrelevant Y220C-TCR. I and J, Tumor growth following ACT of patient 4349’s PBL (1 or 2 × 107 cells) transduced with the R175H-TCR or untransduced (2 × 107 cells). J, Mice injected with either TYK-nu cells or the control 4259 PDX cells were treated with untransduced T cells, R175H-TCR–engineered T cells, or vehicle (PBS). Statistical analyses by two-way ANOVA (D, E, G, I, J) and by log-rank tests (F and H). *, P < 0.05; **, P < 0.01; ***, P < 0.001. A–D were independently repeated at least once.