An mRNA-based workflow for validation of Neo-Epitope Presentation by HLA-I/Peptide Affinity Purification

ABSTRACT: Human leukocyte antigen (HLA) class I restricted neo-epitopes play a key role in inducing an adaptive cytotoxic T lymphocyte response against cancer. To enhance therapeutic and personalized cancer vaccination strategies, the identification of these cancer-specific neo-epitopes is essential. Current workflows for the neo-epitope identification are often laborious and still require computational deconvolution to determine the correct peptide sequence and its corresponding restriction element. In this study, we evaluated the use of an mRNA-based workflow, which allows for more precise purification of HLA-I/peptide (pHLA) complexes and potentially facilitates peptide identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The approach uses mRNA encoding a specific HLA-I molecule linked with a Twin-Strep-Tag (HLA-TST), allowing affinity-based purification and downstream analysis of pHLA. We delivered a proof-of-concept for this approach by co-delivering mRNA encoding a TST-coupled HLA-A*0201 with mRNA encoding the HLA-A*0201-restricted p53 epitope via electroporation in HLA-A*0201- K562 cells. The findings of this study show the promise of the mRNA-based strategy in neo-epitope research. However, further studies are necessary to fully understand the technical factors that can affect the peptides identified by LC-MS/MS.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Cell Culture

SUBMITTER: Kurt Boonen  

LAB HEAD: Karine Breckpot

PROVIDER: PXD062551 | Pride | 2025-06-23

REPOSITORIES: Pride