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An mRNA-based workflow validating neo-epitope presentation through HLA-I/peptide affinity purification.

Arthur Esprit1, Dorien Autaers1, Kris Thielemans1

  • 1Translational Oncology Research Centre, Vrije Universiteit Brussel (VUB), Brussels, Belgium.

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|June 19, 2025
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Summary

This study introduces an mRNA-based workflow to precisely purify and identify human leukocyte antigen (HLA)-peptide complexes. This method aids in validating cancer neo-epitope presentation by antigen-presenting cells (APCs) for personalized cancer vaccines.

Keywords:
IVT-mRNAepitopemass spectrometrypresentationpurification

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Area of Science:

  • Immunology
  • Molecular Biology
  • Cancer Research

Background:

  • Effective cancer vaccines rely on presenting human leukocyte antigen (HLA)-class I-restricted neo-epitopes to cytotoxic T-lymphocytes.
  • Validating neo-epitope presentation by antigen-presenting cells (APCs) is crucial for advancing personalized cancer therapeutics.
  • Current neo-epitope identification methods are often time-consuming and computationally intensive.

Purpose of the Study:

  • To evaluate an mRNA-based workflow for precise purification of HLA-I-peptide (pHLA)-complexes.
  • To facilitate accurate peptide identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • To demonstrate the potential of this workflow in verifying neo-epitope presentation by APCs.

Main Methods:

  • Utilized mRNA encoding a Twin-Strep-Tag (TST) fused to a specific HLA-I molecule (HLA-TST) for affinity-based purification.
  • Co-electroporated mRNA encoding TST-HLA-A*02:01 and an HLA-A*02:01-restricted epitope into HLA-A*02:01-negative APCs.
  • Purified and identified the delivered epitope from pHLA-complexes using LC-MS/MS.

Main Results:

  • Successfully purified pHLA-complexes using the mRNA-based HLA-TST approach.
  • Demonstrated the detection of the delivered epitope via LC-MS/MS.
  • Confirmed the feasibility of verifying neo-epitope presentation in APCs.

Conclusions:

  • The developed mRNA-based workflow offers a precise method for purifying and identifying HLA-peptide complexes.
  • This approach holds significant potential for validating neo-epitope presentation in the context of personalized cancer vaccines.
  • Further research is needed to optimize technical variables for peptide identification by LC-MS/MS.