Therapeutic targeting of the recurrent mutant TP53R248Q neoantigen by T cell receptor-engineered T cells in human cancers

Abstract Background Malignant tumors with TP53 mutations exhibit poor therapeutic outcomes and high recurrence rates. T cell receptor (TCR)-based T cell therapy shows great promise for targeting intracellular cancer neoantigens. However, the immunogenic potential of TP53 hotspot mutations remains poorly characterized. MethodsToidentify HLA-bound TP53 neoantigens, we employed circular mRNA (cmRNA)-encoded tandem TP53 mutants combined with mild acid elution/mass spectrometry (MAE/MS). TP53R248Q-reactive TCR was then isolated from HLA-A*11:01-positive healthy donor via Tetramer-staining and single-cell TCR sequencing. Next, TP53R248Q TCR-T cells functional avidity was assessed via activation markers expression, cytokine secretion, and cytotoxicity assays. The specificity of TP53R248Q TCR-T cells was validated using alanine/glycine substitution assays, structural modeling, and cross-reactivity screens against human proteome-derived peptides. Additionally, in vivo efficacy was evaluated in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models engrafted with TP53R248Q/HLA-A*11:01-positive tumor cells. Results We identified a novel immunogenic neoantigen derived from the recurrent TP53R248Q mutation, presented by the prevalent HLA-A*11:01 allele. Additionally, we isolated a TP53R248Q reactive TCR that specifically recognizes the TP53R248Q mutation without any discernable cross-activity to cognate wild-type TP53 or other TP53 mutants at the same codon position. Functional characterization revealed that TP53R248Q TCR-T cells exhibited selective cytotoxicity against tumor cells expressing both TP53R248Q mutation and HLA-A*11:01 in vitro. Importantly, the adoptive transfer of TP53R248Q TCR-T cells exhibited significant anti-tumor activity in a clinically relevant patient-derived xenograft (PDX) model engrafted with TP53R248Q/HLA-A*11:01-positive human tumor tissues. Conclusions This study validates the immunogenicity of the TP53R248Q hotspot mutation and offers a TCR-based therapeutic potential for TP53R248Q/HLA-A*11:01-positive cancer patients. Given the high mutation frequency of TP53R248Q and the widespread prevalence of HLA-A*11:01 across populations, this treatment has broad clinical potential.