Mass spectrometry based immunopeptidomics is the gold standard for defining the HLA-presented peptide repertoire to the immune system. Discovery immunopeptidomics has led rise to promising targets, some of which have been explored in clinical trials and others contain robust immunological responses such as cytotoxic killing assays. Therefore, there is a plethora of robust peptide targets that have been validated and with the correct modality can be implemented directly for patients in the clinic.
Utilising the advancements in mass spectrometry technology and sensitivity, we employed a Sniper based immunopeptidomics approach leveraging the sensitivity and speed of the ThermoFisher Stellar mass spectrometer. A refined list of previously identified cancer specific epitopes in clinical trials and/or with in vivo T-cell killing assays were clustered by HLA supertypes and curated for scheduled parallel reaction monitoring acquisition. As an initial implementation, we focused on the most prevalent HLA allotype, HLA-A*02:01, selecting 102 peptides to screen rare cancer types with limited immunopeptidomics data, including osteosarcoma, glioblastoma and diffuse midline glioma. To validate our 30-minute assay, we performed rigorous testing and method optimisation with synthetic peptides, Prosit generated in silico libraries and direct DIA acquisition on the Astral mass spectrometer, with method refinement using skyline and PRM conductor. Furthermore, we incorporated 10 housekeeping peptides which were used as internal controls to validate our panel driven PRM approach and act as a quality control for the assay.
This targeted approach robustly identified both shared and tumour specific peptides across the tissues and cell lines. Importantly, actionable antigens were identified from as low as ~1mg of tissue and 1e6 cells. Notable antigens include, PMEL, TP53 and PGK1 and peptides unique to each cancer type. In total, ~20% of these highly immunogenic peptides were identified across each cancer type. All housekeeping peptides were naturally presented and confidently identified, confirming efficient HLA peptide isolation, sensitivity and PRM scheduling fidelity.
Although discovery immunopeptidomics is imperative and remains a critical arsenal to discover clinically relevant antigens, the use of a Sniper Immunopeptidomics approach, with a predefined, HLA-guided panel accelerates the detection of clinically relevant, low-abundant peptides and reduces analysis overhead. Using the sensitivity of the Stellar mass spectrometer in conjunction with method refinement with skyline/PRM conductor, this 30-minute assay can be readily applied for all sample types and can rapidly screen clinical cohorts, informing clinicians possible off the shelf therapeutic avenues to explore.