1239 Uncovering the dark side of the immunopeptidome

Le résumé fourni par la source

Background The immune system targets cancer cells through T cell-mediated recognition of antigenic peptides presented on human leucocyte antigen (HLA) molecules, which is the basis of T cell-based immunotherapies. Their success highly depends on the identification of suitable HLA-presented tumor antigens which are identified by mass spectrometry (MS)-based immunopeptidomics. Substantial efforts have been made to identify naturally presented tumor-associated antigens, including neoepitopes. Only a small fraction of mutations at the DNA level are detected in the immunopeptidome. Similarly, a significant portion of the non-mutated, protein-coding genome remains largely unexplored, as it is either poorly or not detected by MS. These so-called dark spots of the immunopeptidome are either of biological origin or due to technical and methodological limitations of current MS-based approaches.Methods Our recently published immunopeptidomics database 1 containing >9 million HLA class I- and >5 million HLA class II-presented peptides from >5,000 malignant and benign samples was used as a reference for comprehensive mapping of the immunopeptidome dark spot landscape. Alignment analysis of cancer mutations, post translational modifications (PTMs), physiochemical amino acid properties, predicted HLA motifs and presentation processing predictions (pepsickle) were used to uncover the underlying technical, methodological, or biological factors. Moreover, usage of high-sensitive MS technologies combined with hydrophobic desalting steps (restricted access material (RAM), basic reversed-phase fractionation (bRP)) were used.Results Only roughly 30% of the human proteome is covered in recent immunopeptidome data, although a sequence coverage of up to 85-90% is achieved using HLA-binding predictors. Analyses revealed overrepresentation of passenger mutations in dark spots (≈36 mutations per 100 amino acids), highlighting the importance of dark spots for future neoepitope vaccine developments. High-sensitive ion mobility MS could resolve dark spots showing a bias towards peptides rich in acidic amino acids (1143/1959) or highly hydrophobic peptides (575/1959). Combining RAM and bRP, dark spots of 995 highly hydrophobic peptides were resolved. Cell line experiments involving RAM and bRP revealed resolution of 19% of cell line-specific dark spots. Including PTMs such as cysteinylation as variable modifications resolved 258 dark spots, underscoring their significance in immunopeptidomics. Proteasomal cleavage site predictions were performed to investigate potential association with dark spots. These results highlight that hydrophobic peptides or peptides rich in acidic residues as well as PTMs are associated with immunopeptidome dark spots.Conclusions Distinguishing dark spots as either technical and methodological (non-biological), or biological in origin will enhance the accuracy of (neo)epitope selection for T cell-based immunotherapy.Reference Lemke S, Dubbelaar ML, Zimmermann P, Bauer J, Nelde A, Hoenisch Gravel N, Scheid J, Wacker M, Jung S, Dengler A, Maringer Y, Rammensee HG, Gouttefangeas C, Fillinger S, Bilich T, Heitmann JS, Nahnsen S, Walz JS. PCI-DB: a novel primary tissue immunopeptidome database to guide next-generation peptide-based immunotherapy development. J Immunother Cancer 2025 Apr 15;13(4):e011366. doi: 10.1136/jitc-2024-011366. PMID: 40234091; PMCID: PMC12001369.

Ce résumé expose les affirmations des auteurs. BNTIC ne l’interprète pas comme une validation indépendante des résultats.