Non-canonical open reading frame-derived antigens as emerging immune targets in cancer immunotherapy

Background/Objectives Cancer immunotherapy predominantly targets mutation-derived neoantigens, thereby limiting its efficacy in low-mutational-burden tumours such as pancreatic and paediatric cancers. Non-canonical open reading frames (ncORFs) generate a diverse repertoire of peptides, the dark immunopeptidome, including upstream ORFs, lncRNA-derived ORFs, and transposable element-derived ORFs, which are efficiently presented by MHC class I molecules. Methods A narrative synthesis of peer-reviewed literature integrating ribosome profiling, immunopeptidomics, proteogenomics, and functional T-cell assays was conducted across multiple tumour types. Results Evidence demonstrates that ncORF-derived peptides are frequently tumour-enriched at the transcript or translation level, and a proportion has been directly identified at the peptide level by HLA-elution immunopeptidomics from primary tumour tissue; a subset can prime antigen-specific CD8+ T-cell responses ex vivo, with cytotoxic activity demonstrated against peptide-pulsed targets in vitro and, in selected cases, against endogenously processed tumour cells. These ncORF-derived antigens may partially evade, and direct human thymic immunopeptidome data are lacking. Conclusions Incorporating ncORF-derived antigens into personalised cancer vaccine strategies could expand therapeutic targets, especially for low-mutational burden and therapy-resistant tumours, but requires further bioinformatic refinement and rigorous safety validation.