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. We and others have recently shown that a high proportion of all presented peptides are generated through proteasome-mediated splicing of non-contiguous regions of proteins to form novel “spliced” peptide antigens.
We have developed a novel workflow for the identification of cis (donor segments from same antigen) and trans-spliced peptide antigens (donor segments from two different antigens). We have applied our approach to HLA-bound peptides derived from multiple cancer cell lines including melanoma, triple-negative breast cancer, mesothelioma, DIPG, colon cancer as well as well-established murine models of breast cancer, mesothelioma and melanoma. We found 20-30% of all peptides presented by HLA-class-I molecules are derived from post-translational splicing. Of note, in each sample 100-300 of these spliced peptides were derived, from cancer-associated antigens (CAA) engendering cancer specificity of these spliced peptides. We have confirmed the authenticity of a series of CAA-derived spliced peptides using retrospectively synthesised peptides. Moreover, immunogenicity studies of a subset of the CAA-derived peptides highlighted that several of these peptides were immunogenic in multiple HLA-matched patients. Vaccination of mice with some of the cancer-specific spliced peptides significantly reduced the size of tumors and clinical studies for vaccination of melanoma patients with some of discovered cancer-specific spliced peptides are ongoing.
These observations highlight the breadth and complexity of the repertoire of immunogenic peptides available for exploitation therapeutically and suggest that spliced peptides may be a major class of tumour antigens. Indeed, we found spliced peptides may yield more immunogenic epitopes than genome template cancer peptides and, in the absence of high-affinity CAA-derived HLA ligands, can generate higher affinity neoepitopes for interaction with host HLA allomorphs. Understanding the nature and abundance of spliced peptides has high relevance for discovery of novel targets of T cell immunity and will have significant implications for further immunotherapeutic approaches in cancer.