Background: The proteasome is a multi-subunit protease that degrades intracellular proteins into short peptides, which are then presented on the cell surface in complex with Human Leukocyte Antigen (HLA) molecules to T-cells. This HLA-bound peptide repertoire is termed the immunopeptidome. These peptides conventionally derive from contiguous sequences of proteins, yet recent studies have identified that a proportion can result from splicing of non-contiguous regions of the same or different proteins. Such spliced peptides have also been identified as being immunogenic in human cancers. Whilst most studies suggest a predominant role of the proteasome in catalysing peptide splicing, other proteases crucial to antigen presentation may also contribute to the spliced immunopeptidome. Endoplasmic reticulum aminopeptidase (ERAP) is a protease that trims HLA-bound peptides at their N-terminus. Studies have shown that its inhibition leads to an increase in the presentation of immunogenic peptides, but the extent to which this inhibition affects peptide splicing remains unexplored. This study has utilised a novel ERAP inhibitor on patient-derived melanoma cell lines to investigate its impact on the immunopeptidome and identify potential novel peptide targets for T-cell immunotherapy.
Methods: This study combines peptide sequencing performed by data-independent acquisition (DIA) mass spectrometry with PEAKS Studio software and novel bioinformatics algorithms to identify and quantify linear and spliced peptides presented by patient-derived melanoma cells under normal and ERAP1 inhibitor-treated conditions (500ng/ml and 2μg/ml; novel ERAP1 inhibitor from Grey Wolf Therapeutics).
Results: We generated a spectral library of ~120,000 peptides from replicates of immunopeptidomics data derived from patient-derived melanoma cell lines. This library was then used to interrogate DIA data, which revealed ~5000-11000 unique peptides quantified per cell line. Spliced peptides made up between 23-34% of each immunopeptidome. ERAP1 inhibition led to a remodelling of the immunopeptidome with only ~20% overlap between peptides presented under untreated and inhibited conditions, an increase in 10-12mer peptides, and an overall decrease in the proportion of spliced peptides by ~7%. Importantly, inhibition of ERAP led to a significant increase in the presentation of peptides derived from melanoma-associated antigens (MAA).
Conclusion: In this study, we observed that ERAP1 inhibitor treatment led to significant changes in the HLA-bound immunopeptidome. The repertoire of ERAP inhibited peptides contains significantly more MAA derived peptides as compared to the conventional peptidome. ERAP1 inhibition also led to a decrease in the proportion of spliced peptides, which shows the potential role of ERAP in splicing. These results suggest that inhibition of non-proteasomal proteases can change the immunopeptidome creating potentially novel targets for T-cell immunotherapy.