Abstract 5838: Quantification of ERAP-1 inhibition-dependent HLA-I peptides using targeted mass spectrometry for TCR-based therapeutics target discovery

Abstract Human leukocyte antigens (HLA) play a central role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. The identification of neoantigens, unique to individual tumors due to somatic mutations, has become a focal point in immunopeptidomics studies. In particular, accurate quantification of immunogenic peptides to evaluate their potential as novel targets for TCR-based therapeutics has been of increasing interest in immuno-oncology and autoimmune diseases, where regulating the immune response is of growing therapeutic importance. Additionally, precise (neo)antigen quantification can guide the development of personalized vaccines and cell therapies, further highlighting the need for robust methods to evaluate peptide presentation in diverse disease contexts. Current methodologies to measure presented antigens are hampered by low sensitivity. Here, we employed a targeted mass spectrometry technique FAIMS-PRM to overcome these challenges. This method allows for development of highly sensitive targeted assays for the quantification of immunopeptide(s) of interest from minimal sample inputs: 25 million cells or 15mg fresh frozen tissue. Coupled to the spike-in of chemically synthesized internal standards (SIS), FAIMS-PRM enabled the estimation of copy number per cell of immunopeptides, thus providing essential information to assess potential targets in both disease and healthy matrices. To exemplify the analytical benefits of this workflow, we quantified neoantigens presented by HLA-I complexes in cancer cells upon treatment with a small molecule ERAP1 inhibitor. ERAP1 trims peptide precursors to optimal lengths for loading onto HLA-I molecules in the endoplasmic reticulum, thereby regulating the repertoire of peptides available for recognition by CD8+ T cells. Inhibition of ERAP1 results in an entirely novel repertoire of peptides being presented for immune recognition, generating functional anti-tumor T-cell responses and a novel target space for TCR-based therapies. In this study, a panel of peptide assays containing 30 ERAP1 inhibition-dependent HLA Class I peptides was developed with SIS for each target. This includes peptides from industry-validated TCR targets and from novel targets selected from prior discovery studies. In the tested melanoma and ovarian cancer cell lines, this targeted assay could demonstrate a lower limit of quantification (LLOQ) reaching low single-digit range (1-3 copies) per cell. Treatment of these cells with ERAP1 inhibitors resulted in a 2- to 10-fold increase in epitope copy number depending on cell type. In conclusion, we established a robust pipeline for HLA class I peptide quantification which can be routinely applied to estimate copy number per cell in biological samples such as cancer cell lines or tissues for target discovery. Citation Format: Anamarija Pfeiffer, Lucy Yang, Maik Mueller, Daniel Redfern, Yuehan Feng, Kris Clark, James Bishop, Juanita Carrey, Sam Humphrey, Wayne Paes. Quantification of ERAP-1 inhibition-dependent HLA-I peptides using targeted mass spectrometry for TCR-based therapeutics target discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5838.