Quantification of ERAP-1 Inhibition-Dependent HLA-I Peptides Using Targeted Mass Spectrometry for TCR-Based Therapeutics Target Discovery

Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays an important role in shaping the immunopeptidome across all human cells by trimming peptide precursors prior to loading onto major histocompatibility complex class I (MHC-I) molecules in the endoplasmic reticulum (ER). This process modulates the repertoire of peptides available for recognition by CD8+ T cells, directly influencing immune surveillance. Therapeutic inhibition of ERAP1 modulates neoantigen repertoire driving the presentation of different cancer antigens, thereby triggering functional anti-tumor T-cell responses and opening new target spaces for TCR-based therapies.

To study immunopeptides bound to MHC-I or MHC-II, discovery mass spectrometry (MS) is essential for unbiased identification of the presented antigen repertoire. While discovery MS is typically semi-quantitative, precise characterization of antigens of interest requires accurate estimation of copy number per cell, which provides valuable insights into antigen abundance and potential immunogenicity. However, current methodologies for measuring these antigens are limited by low sensitivity, making it challenging to quantify immunopeptides from complex biological samples.