ArticleFrontiers in immunology2025
An mRNA-based workflow validating neo-epitope presentation through HLA-I/peptide affinity purification.
Article in Frontiers in immunology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Presentation of human leukocyte antigen (HLA)-class I-restricted neo-epitopes is key for inducing an adaptive cytotoxic T-lymphocyte response against cancer. Validating presentation of these cancer-specific neo-epitopes following delivery to antigen-presenting cells (APCs) is critical to advance personalized therapeutic cancer vaccines. Current workflows for neo-epitope identification are often laborious and depend on computational deconvolution to determine the correct peptide sequence and its corresponding restriction element. We evaluated an mRNA-based workflow for more precise purification of HLA-I-peptide (pHLA)-complexes, facilitating peptide identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach uses mRNA encoding a specific HLA-I-molecule fused to a Twin-Strep-Tag (HLA-TST), allowing affinity-based purification and downstream analysis of pHLA-complexes. As a proof-of-concept, we co-electroporated mRNA encoding TST-HLA-A*02:01 and mRNA encoding an HLA-A*02:01-restricted epitope in HLA-A*02:01-negative APCs. We demonstrated successful purification and detection of the delivered epitope via LC-MS/MS. These findings highlight the potential of the mRNA-based workflow to verify neo-epitope presentation by APCs. Still, further investigation is necessary to fully understand the technical variables that can influence peptide identification by LC-MS/MS.
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