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Abstract Circular RNAs (circRNAs) are covalently closed non-coding RNAs lacking the 5’ cap and the poly-A tail. Nevertheless, it has been demonstrated that certain circRNAs can undergo active translation. Therefore, aberrantly expressed circRNAs in human cancers could be an unexplored source of tumor-specific antigens, potentially mediating anti-tumor T cell responses. This study presents an immunopeptidomics workflow with a specific focus on generating a circRNA-specific protein fasta reference. The main goal of this workflow is to streamline the process of identifying and validating human leukocyte antigen (HLA) bound peptides potentially originating from circRNAs. We increase the analytical stringency of our workflow by retaining peptides identified independently by two mass spectrometry search engines and/or by applying a group-specific FDR for canonical-derived and circRNA-derived peptides. A subset of circRNA-derived peptides specifically encoded by the region spanning the back-splice junction (BSJ) are validated with targeted MS, and with direct Sanger sequencing of the respective source transcripts. Our workflow identifies 54 unique BSJ-spanning circRNA-derived peptides in the immunopeptidome of melanoma and lung cancer samples. Our approach enlarges the catalog of source proteins that can be explored for immunotherapy.

The immunogenicity of peptides is believed to be determined by their high-affinity binding to MHC I. Here authors show that low-affinity MHC I-peptide interactions are also able to trigger robust T cell response and anti-tumour immunity in vivo.

Non-canonical HLA-bound peptides from presumed non-coding regions are potential targets for cancer immunotherapy, but their discovery remains challenging. Here, the authors integrate exome sequencing, transcriptomics, ribosome profiling, and immunopeptidomics to identify tumor-specific non-canonical HLA-bound peptides.