The Dark Matter of the Immunopetidome: Cryptic Peptides as Tumor Antigens in Melanoma.

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Malignant melanoma is the deadliest form of skin cancer and contributes the majority of skin cancer-related mortality worldwide. Immunotherapies called immune checkpoint inhibitors (ICI) have shown significant activity in patients with advanced melanoma. ICI unleashes and induces T cell mediated anti-tumor immunity and is now broadly used in modern oncology to treat human malignancies. Activity of ICI is associated with a high load of somatic mutations, which give rise to so-called neoantigens. Recently, neoantigens were detected directly in samples from melanoma metastases by mass spectrometry (MS). However, the frequency of these mutated antigens among the sum of all HLA peptides (the immunopeptidome) seems to be lower than indicated by in silico predictions. In contrast, T cell responses against cryptic peptides are frequent in patients with metastatic melanoma, but identification of these peptides by MS is challenging since they cannot be found in protein databases. De novo sequencing algorithms can determine peptide sequences directly from MS/MS without using databases. By using de novo sequencing, we were able to identify up to 20% of cryptic peptides within the HLA immunopeptidome of tumor samples. Parts of these cryptic peptides originate from translation of non-canonical open reading frames or non-coding regions (e.g. 5‘-UTRs or ncRNAs). The origin of the majority of cryptic peptides, however, remains unknown. Our preliminary data indicate that a substantial fraction of the human immunopeptidome consists of cryptic peptide. Since 99% of all somatic mutations in cancer affect non-protein coding regions of the genome, we hypothesize that cryptic peptides might be enriched for (mutated) neoantigens. In this project, we want to combine whole genome sequencing, RNAseq (RiboMinus), MS and de novo sequencing to systematically assess the origin and specificity of cryptic HLA peptides in melanoma tissue. If there are cryptic peptides exclusively found in tumor tissue, these antigens might offer a unique and promising way to design personalized immunotherapies.