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Background Mutant forms of p53 are present in most human cancers, but targeting these proteins remains a major challenge. The recent identification of immunogenic neoantigens originating from mutations at TP53 R175H has enabled personalized immunotherapy strategies applicable to approximately 2.6% of all cancer patients.1 CAR T and bispecific antibody therapies targeting the public neoantigen HMTEVVRHC, derived from p53R175H, have already demonstrated robust pre-clinical efficacy.2–4 Here, we assess HMTEVVRHC as an off-the-shelf vaccine candidate by generating altered peptide ligands (APLs) that enhance antigen presentation and T cell priming while preserving TCR cross-reactivity.Methods Candidate APLs were generated by mutating anchor residues at both N- and C-terminal sites within the peptide to enhance cognate HLA-A*02:01 binding and TCR recognition. Candidate APLs were selected based on netMHC4.0 and PyRosetta FlexPepDock predictions. Tap-deficient T2 cells were pulsed with APLs to assess HLA-A*02:01 stability by flow cytometry. To functionally assess antigen presentation by APL-pulsed T2 cells, we measured IFNγ production in T cell co-cultures in the presence of H2-ScDb, a bispecific antibody against the HMTEVVRHC/HLA-A*02:01 complex. 2 TCR cross-reactivity between the parent peptide and APLs was assessed with a panel of patient-derived TCRs, either transfected into Jurkat NFAT reporter cells or by CRISPR knock-in of peripheral blood mononuclear cells. To determine the capacity of each APL to elicit robust T cell responses in vivo, HLA-A*02:01 transgenic mice received a 21-day prime and boost prophylactic immunization consisting of synthetic long peptides derived from each APL. Splenocytes were pulsed ex vivo with each respective APL and unmodified parent variant. The percentage of antigen-specific CD8 T cells was measured through flow cytometry of intracellular IFNγ staining.Results APLs derived from HMTEVVRHC elicited up to a 10-fold increase in HLA-A*02:01 binding affinity and extended the peptide-HLA half-life by up to 40 hours. T cell IFNγ secretion doubled when co-cultured with T2 cells pulsed with each APL in the presence of H2-ScDb. Patient TCRs showed remarkable cross-reactivity and increased binding affinity. In vivo vaccination with each APL caused an increase in antigen-specific T cell priming compared to the unmodified parent. Consistent with the in vitro studies, the majority of the expanded T cells were cross-reactive to the original epitope.Conclusions Altered peptide ligands derived from a common p53 neoantigen promote superior immunogenicity compared to the unmodified parent peptide. This strategy for optimizing a public neoantigen may have broad applications in neoantigen peptide design and cancer vaccine development.References Kim S, et al. Adoptive cellular therapy with autologous tumor-infiltrating lymphocytes and T-cell receptor-engineered T cells targeting common p53 neoantigens in human solid tumors. Cancer Immunol Res. 2022;3;10(8):932–946.Hsiue E, et al. Targeting a neoantigen derived from a common TP53 mutation. Science. 2021;371:6533.Mog B, et al. Preclinical studies show that Co-STARs combine the advantages of chimeric antigen and T cell receptors for the treatment of tumors with low antigen densities. Sci Transl Med. 2024;16:755.Daosheng H, et al. TCR-mimicking STAR conveys superior sensitivity over CAR in targeting tumors with low-density neoantigens. Cell rep. 2024;11:44.