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Immunotherapies, such as immune checkpoint inhibitors (ICIs), have revolutionized management of some cancers but have little benefit for microsatellite-stable colorectal cancer patients (MSS-CRC). This is, in part, due to the low mutations and neoantigen expression in this immunogenically "cold" MSS-CRC. Therefore, we aim to develop novel shared neoantigen-based therapeutic cancer vaccine to reinvigorate antitumor immunity and enhance the therapeutic benefit of radiotherapy in MSS-CRC. To identify novel highly expressed and shared neoantigens, we collected 40 match-paired adjacent normal and tumor tissues from MSS-CRC patients for WES-seq, RNA-seq, and liquid chromatography–MS/MS (LC-MS/MS). By incorporating these databases, we established Neoantigen Discovery and Validation (NeoDiva) system to identify a cluster of highly expressed and shared neoantigens derived from non-coding regions and evaluate its immunogenicity by HLA-A*11 transgenic mice. We then develop a neoantigen-based therapeutic cancer vaccine by an engineered adenovirus-associated virus (AAV) to evaluate its therapeutic efficacy in combination with radiotherapy in MSS-CRC animal model. We identified a cluster of highly expressed and shared neoantigens (HLA-A*11-restricted) derived from non-coding regions. The immunogenicity of these novel neoantigens was demonstrated by HLA-A*11 transgenic mice and ex vivo stimulation. Moreover, the engineered AAV-based neoantigen cancer vaccine significantly eradicates cancer cells, prevents distant metastasis, prolong survival period in combination with radiotherapy. By flow cytometry, ELISPOT and MHC-I-tetramer assay, we demonstrated the recruitment of tumor-infiltrating lymphocytes was remarkably increased and neoantigen-specific T cell response was enhanced. Moreover, these isolated neoantigen-specific T cells can recognize cancer cells and produce IFNg to kill cancer cells. Neoantigens identified by our NeoDiVa platform, via the combination of radiotherapy and a novel AAV vaccine delivery system, boosted antigen-specific T-cell function and improve tumor control of limnologically "cold" MSS colorectal cancer in vivo. We are in the process of obtaining an IND and initiating Phase I/II clinical trial to validate safety and efficacy of these exciting findings. [ABSTRACT FROM AUTHOR]