Tumor-Specific CircRNA-Derived Antigen Peptide Identification for Hepatobiliary Tumors

The application of tumor antigen-based immunotherapy is hindered by the rarity of validated immunogenic peptides. In this study, we aimed to investigate the potential of circular RNAs (circRNAs) as a novel source of tumor antigen peptides in hepatobiliary tumor organoids. Using RNA-sequencing (RNA-seq) with an algorithm-based score tool, 3950 translated tumor-specific circRNAs were predicted to generate 18 971 antigen peptides in 27 organoids. In view of the antigen landscape, 11 amino acid length (mer) peptides and human leukocyte antigen (HLA)-A binding peptides harbored the highest immunogenicity-related scores. In three out of five analyzed organoids, 13 predicted antigen peptides were directly confirmed as HLA-A, -B, and -C (HLA-ABC) binding peptides with mass spectrometry (MS)-based immunopeptidomics. CircRNA-derived tumor-specific peptides presented by the HLA-ABC molecules stimulated cluster of differentiation 8 (CD8) T cells to exhibit increased CD107a interferon γ (IFNγ) co-expressions and IFNγ secretion in flow cytometry and enzyme-linked immunosorbent assay (ELISA). Cytotoxic T cell activity targeting the organoids, induced by the immunogenic circRNA-derived peptides, was verified in a killing assay. Notably, the antigen peptide YGFNEILKK from circTBC1D15 was not only recognized as an HLA-ABC-presented peptide of the organoids but also drastically reduced the tumor organoid survival rate. Our findings highlight a crucial subset for generating tumor antigens, which has implications for targeting tumor-specific circRNAs in cancers.