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Therefore, the establishment of a solid pipeline for the downstream selection of clinically relevant peptides and the subsequent creation of therapeutic cancer vaccines are of utmost importance. Indeed, the use of peptides for eliciting specific antitumor adaptive immunity is hindered by two main limitations: the efficient selection of the most optimal candidate peptides and the use of a highly immunogenic platform to combine with the peptides to induce effective tumor-specific adaptive immune responses. Here, we describe for the first time a streamlined pipeline for the generation of personalized cancer vaccines starting from the isolation and selection of the most immunogenic peptide candidates expressed on the tumor cells and ending in the generation of efficient therapeutic oncolytic cancer vaccines. This immunopeptidomics-based pipeline was carefully validated in a murine colon tumor model CT26. Specifically, we used state-of-the-art immunoprecipitation and mass spectrometric methodologies to isolate &gt;8000 peptide targets from the CT26 tumor cell line. The selection of the target candidates was then based on two separate approaches: RNAseq analysis and HEX software. The latter is a tool previously developed by Jacopo, 2020, able to identify tumor antigens similar to pathogen antigens in order to exploit molecular mimicry and tumor pathogen cross-reactive T cells in cancer vaccine development. The generated list of candidates (26 in total) was further tested in a functional characterization assay using interferon-\u03b3 enzyme-linked immunospot (ELISpot), reducing the number of candidates to six. These peptides were then tested in our previously described oncolytic cancer vaccine platform PeptiCRAd, a vaccine platform that combines an immunogenic oncolytic adenovirus (OAd) coated with tumor antigen peptides. In our work, PeptiCRAd was successfully used for the treatment of mice bearing CT26, controlling the primary malignant lesion and most importantly a secondary, nontreated, cancer lesion. These results confirmed the feasibility of applying the described pipeline for the selection of peptide candidates and generation of therapeutic oncolytic cancer vaccine, filling a gap in the field of cancer immunotherapy, and paving the way to translate our pipeline into human therapeutic approach.<\/jats:p>","DOI":"10.7554\/elife.71156","type":"journal-article","created":{"date-parts":[[2022,3,16]],"date-time":"2022-03-16T14:15:12Z","timestamp":1647440112000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.71156","source":"Crossref","is-referenced-by-count":38,"title":["A novel immunopeptidomic-based pipeline for the generation of personalized oncolytic cancer vaccines"],"prefix":"10.7554","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4012-4310","authenticated-orcid":true,"given":"Sara","family":"Feola","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/040af2s02","id-type":"ROR","asserted-by":"publisher"}],"name":"Drug Research Program (DRP) 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