A peptide-signal amplification strategy for the detection and validation of neoepitope presentation on cancer biopsies

Targeting the right cancer-specific peptides presented by Human Leukocyte antigen (HLA) class I and II molecules on the tumour cell surface is a crucial step in cancer immunotherapy. Numerous approaches have been proposed to predict the presentation of potential neoepitopes that may be targeted through various immune-based therapies. Often based on patient specific somatic mutations, the routine validation of their actual appearance on the tumour cell surface is a significant barrier to realising personalised cancer immunotherapy. This can be attributed to the lack of robust and adaptable assays for antigen presentation that offer the required sensitivity to deal with the often very limited amounts of available patient tumour tissue. Rather than personalise individual assays we propose the use mass spectrometry to identify tumour neoepitopes from immunoprecipitated HLA-bound peptides directly isolated form the surface of tumour biopsies. We have developed a microscale HLA-peptide complex immunoprecipitation protocol combined with tandem mass tagging (TMT) to directly sequence HLA-bound peptides using mass spectrometry. Using this strategy, we identified HLA-bound peptides from as few as ~1000 cultured cells and from a small piece (~1 mg) of whole melanoma tumour tissue, encompassing epitopes derived from Melanoma-associated antigens and potential neoantigens.