Neoantigen screening identifies broad TP53 mutant immunogenicity in patients with epithelial cancers

Parisa Malekzadeh, Anna Pasetto, Paul F Robbins, Maria R Parkhurst, Biman C Paria, Li Jia, Jared J Gartner, Victoria Hill, Zhiya Yu, Nicholas P Restifo, Abraham Sachs, Eric Tran, Winifred Lo, Robert Pt Somerville, Steven A Rosenberg, Drew C Deniger, Parisa Malekzadeh, Anna Pasetto, Paul F Robbins, Maria R Parkhurst, Biman C Paria, Li Jia, Jared J Gartner, Victoria Hill, Zhiya Yu, Nicholas P Restifo, Abraham Sachs, Eric Tran, Winifred Lo, Robert Pt Somerville, Steven A Rosenberg, Drew C Deniger

No abstract available

Keywords: Cancer; Immunology; Oncology; P53; T cells.

Conflict of interest statement

Conflict of interest: PM, AP, PFR, MRP, WL, SAR, and DCD have a patent application (no. 62/565,383) for TCRs described in this manuscript. PM, SAR, and DCD have a patent application (no. 62/565,464) for methods described in this manuscript.

Figures

Figure 1. TP53 hot spot mutations are immunogenic and elicit intratumoral T cell responses to p53 neoantigens.

(A) Patients with a tumor expressing a TP53 mutation. (B) Classification of TP53 mutations from resected tumors. (C) The overall frequencies of each missense mutation in TP53 from all patients sequenced by p53 codon where the specific amino acid change frequency is given for selected mutations. (D) Screening results from patient 9 as measured by IFN-γ ELISPOT. (E) Expression of 41BB from patient 9’s TIL culture 1 in response to TP53 TMGs. (F) Overall frequency of TIL responses to mutated TP53, which include TMG, peptide, or both (Supplemental Table 5). (G) Frequencies of verified positive TIL responses from selected fragment cultures (some patients had >1) to p53 R175H, Y220C, G245S, R248Q, R248W, and R282W neoantigens. The upregulation of 41BB minus the background in response to mutated p53 peptide (CD4) or TMG (CD8) is reported. Types of T cell responses can be found in Table 1.

Figure 2. Immunogenic TP53 hot spot mutations could be restricted by common HLA alleles and targeted by TCRs isolated from p53 neoantigen–reactive TILs.

(A) HLA class I restriction of p53Y220C (patient 5, TIL culture 4259-F1) and p53R248W (patient 9, TIL culture 4266-F1) neoepitopes. Data are mean ± SEM, n = 3 technical replicates. > indicates 1,250 pg/ml IFN-γ. (B) Peptide mapping of CD4+ T cell responses from patient 4 to p53R175H neoepitopes. (C) HLA class II restriction for p53Y220C neoantigen from patient 5. (D) HLA allele frequencies from selected populations (http://www.allelefrequencies.net/default.asp). (E) Specificity of HLA-DRB*13:01/p53R175H–reactive TCR to 25 amino acid p53R175H peptides.

Figure 3. Tumor cells naturally process and present p53 neoantigens to T cells.

(A) Expression of 41BB on CD8+ T cells expressing HLA-A*68:01/p53R248W neoantigen–specific TCR following coculture with autologous tumor cell line (TC), which was incubated with anti-HLA class I or II blocking antibodies or p53R248W peptide. (B) Intracellular cytokine staining of cocultures of CD8+ T cells expressing HLA-A*02:01/p53R175H neoantigen–specific TCR and Saos2 cells alone (HLA-A*02:01+ and p53NULL; gray circles on left graph or gray dots on right plot) or overexpressing p53R175H full-length protein (red squares on left graph or red dots on right plot). (C) Secretion of IFN-γ in cocultures of HLA-A*02:01/p53Y220C neoantigen–specific T cells (CD8+-enriched TIL culture 4259-F1 from patient 5) with HLA-A*02:01–positive tumors from unrelated donors and Saos2 tumor cells overexpressing full-length p53Y220C protein. > indicates 1,250 pg/ml IFN-γ. Data in panels B and C are mean ± SEM, n = 3 technical replicates, and 2-tailed Student’s t tests were performed for statistical analyses (**P < 0.01 and ***P < 0.001).