Antigen-recognition by CD8+ T cells is governed largely by the pool of peptide antigens presented on the cell surface in the context of HLA class I complexes. Recent studies have shown not only a high degree of plasticity in the immunopeptidome, but also that a considerable fraction of all presented peptides are generated through proteasome-mediated splicing of non-contiguous regions of proteins to form novel peptide antigens.
We have developed a novel workflow for the identification of cis and trans-spliced peptide antigens. We have applied our approach to p-HLA derived from multiple melanoma cell line and identified~ 30% cis and trans HLA-I spliced peptides . Of note, more than more 100 spliced peptides were derived from melanoma-associated antigens (MAA) and ~40% of known MAA were only represented by spliced peptides. We have confirmed the authenticity of a series of MAA spliced peptides by corresponding synthetic peptides. Moreover, immunogenicity studies of a subset of the MAA shown that several of these peptides were shown to be immunogenic in unrelated melanoma patients.
These observations highlight the breadth and complexity of the repertoire of immunogenic peptides that may be exploited therapeutically and suggest that spliced peptides may be a major class of tumour antigens. We found spliced peptides may yield more immunogenic epitopes than are available from the viral/cancer genome. Moreover, some antigens lack high-affinity HLA-ligands and the peptide splicing mechanism can generate higher affinity neoepitopes for interaction with host HLA allomorphs. Understanding the nature and abundance of spliced peptides has a high relevance for our understanding of potential novel targets of T cell immunity and will have significant implications for further immunotherapeutic approaches.