At least two tumors per group were collected and minced into pieces of 1C2?mm of diameter using a sterile scalpel. by all Gram-negative bacteria and, thanks to their plasticity and unique adjuvanticity, are emerging as an attractive vaccine platform. To test the applicability of OMVs in cancer immunotherapy, we decorated them with either one or two protective epitopes Ly6a present in the B16F10EGFRvIII cell line and tested the protective activity of OMV immunization in C57BL/6 mice challenged with B16F10EGFRvIII. Materials and methods The 14 amino acid B cell P505-15 (PRT062607, BIIB057) epitope of human epidermal growth factor receptor variant III (EGFRvIII) and the mutation-derived CD4+ T cell neo-epitope of gene (B16-M30) were used to decorate OMVs either alone or in combination. C57BL/6 were immunized with the OMVs and then challenged with B16F10EGFRvIII cells. Immunogenicity and protective activity was followed by measuring anti-EGFRvIII antibodies, M30-specific T cells, tumor-infiltrating cell population, and tumor growth. Results Immunization with engineered EGFRvIII-OMVs induced a strong inhibition of tumor growth after B16F10EGFRvIII challenge. Furthermore, mice immunized with engineered OMVs carrying both EGFRvIII and M30 epitopes were completely protected from tumor challenge. Immunization was accompanied by induction of high anti-EGFRvIII antibody titers, M30-specific T cells, and infiltration of CD4+ and CD8+ T cells at the tumor site. Conclusion OMVs can be P505-15 (PRT062607, BIIB057) decorated with tumor antigens and can elicit antigen-specific, protective antitumor responses in immunocompetent mice. The synergistic protective activity of multiple epitopes simultaneously administered with OMVs makes the OMV platform particularly attractive for cancer immunotherapy. Keywords: bacterial outer membrane vesicles, cancer immunotherapy, EGRRvIII, cancer neoepitopes, BALB/c-CT26 cancer mouse model, precision medicine Introduction All cancer therapies attempt to exploit the differences existing between tumor and normal cells. Since our immune system is built to target and destroy the non-self, theoretically cancer vaccination is the safest, most natural, and effective therapeutic approach against cancer. Indeed, a large number of preclinical and clinical studies involving cancer vaccines have been described over the last two decades. Unfortunately, in the clinical settings, the results so far have been disappointing. Klebanoff et al. (1) reported a cumulative analysis of several vaccine trials run from 2004 to 2009 and included 936 patients with different types of solid tumors. Using response rate as a measure of positive outcome, the conclusion of the study was that only 3.6% of the patients had an objective benefit from vaccination. The authors concluded that for cancer vaccines to become effective the strategies so far used for their formulation need be substantially revisited. An ideal cancer vaccine should include three elements: (1) a cocktail of tumor-specific and/or tumor-associated antigens (TSA/TAAs), (2) one or more potent immune-stimulatory molecules (adjuvants), and (3) a delivery system which allows the co-delivery of cancer antigens and adjuvant(s) to antigen presenting cells (APCs). The absence of just one of these elements can make the vaccine incapable of counteracting the corrupted tumor microenvironment (containing regulatory T cells and aberrantly matured myeloid cells), and the highly mutable tumor targets (driving antigen loss and immune evasion). Enthusiasm for therapeutic cancer vaccines has been recently rejuvenated by two major discoveries. First, it has been shown that the large number of mutations occurring in most tumors (2) creates neo-epitopes, which can become the targets of both CD4+ and CD8+ T cells. Neo-epitope-specific T cells have been found among tumor-infiltrating lymphocytes (TILs), and when amplified from tumor biopsies and introduced back into patients, TILs can exert antitumor activities (3). Moreover, the impressive therapeutic effect of checkpoint inhibitor antibodies observed in a fraction of patients has been shown to correlate with the number of tumor-associated mutations (4C6). Consequently, vaccines formulated with neo-epitopes have recently been created and shown to be highly effective in preventing tumor growth P505-15 (PRT062607, BIIB057) in different preclinical settings (7). Second, Kranz and coworkers (8) have demonstrated that when administered intravenously (i.v.) in melanoma patients, negatively charged liposomes carrying TSA encoding synthetic RNAs were efficiently taken up by splenic DCs, resulting in a potent elicitation of TAA-specific CD4+ and CD8+ T.