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Abstract

Persistent infection with high-risk types of Human Papillomavirus (HPV), particularly HPV16, is a significant factor in the development of cervical cancer (CxCa) and a subset of head and neck squamous cell carcinoma (HNSCC), namely oropharyngeal cancer. The current standard first-line treatment for advanced cases of both cancers involves cisplatin-based chemoradiotherapy combined with surgery. While this approach has improved survival rates since implementation, relapse still occurs in up to 30% of CxCa patients. Moreover, although HPV-positive HNSCC patients generally have a more favorable prognosis compared to those with HPV-independent disease, approximately 20% still experience disease progression. These observations motivate the research for a more specific therapeutic strategy. The ideal targets for a promising immunotherapeutic approach are HPV oncoproteins E6 and E7, key players in cellular transformation and immortalization by disruption of the normal cell cycle regulation and inhibition of apoptosis. These foreign proteins, consistently present in HPV-driven cancer cells, are processed into peptides that form part of the cancer cell immunopeptidome – the collection of HLA-presented peptides. The overall aim of the Division of Immunotherapy & Immunoprevention is to capitalize on these features and develop a therapeutic HPV vaccine. To that end, we utilize immunopeptidomics, a liquid chromatography-mass spectrometry (LC-MS) based method that enables identification of HLA-presented epitopes. Specifically, a targeted LC-MS technique capable of detecting low-abundance epitopes, including those derived from HPV and mutation-derived neoepitopes, was developed in the group. In this research, I aimed to investigate potential effects of chemoradiation on the immunopeptidome of treated versus untreated CxCa and HNSCC cell lines, as immunomodulatory effects of chemoradiotherapy are reported. Therefore, standard treatment regimens were simulated in vitro using clinically relevant combinations of cisplatin and radiation while maintaining a surviving fraction of CxCa and HNSCC cell lines. HLA surface levels, E6 and E7 expression, and LC-MS peptide identifications were compared across four experimental groups. Furthermore, to seek potential neoepitopes arising from mutations, and to attest their presence after the treatment, whole exome sequencing and RNA sequencing of all cell fractions were performed, followed by validation of the resulting neoepitope candidates using our targeted LC-MS approach. This work resulted in identification of both HPV16-derived peptides conserved post-treatment and putative neoepitopes. These findings will contribute to the development and strategic implementation of an effective therapeutic HPV vaccine. Additionally, through LC-MS method development, the study branched into a multilevel characterization of a CxCa cell line previously considered HPV-independent that resulted in genomic, translational and functional evidence of the cell line´s HPV16 transformation.