Cancer Testis Antigens (CTAs) are a large family of tumor-associated antigens (TAAs) expressed in human tumors of different histological origin, but not in normal tissues, thus representing ideal targets for tumor-specific immunotherapeutic approaches. Based on the knowledge that CTA expression is regulated at epigenetic level, we developed a novel vaccine consisting of autologous antigen-presenting cells and other cellular components able to concomitantly express CTA for a prolonged time. In detail, the vaccine is generated from peripheral blood mononuclear cells (PBMC), ex vivo activated and in vitro treated with DNA hypomethylating agents (DHA) to induce/up-regulate a simultaneous expression of multiple methylation-sensitive CTA. In this research project, we undertook pre-clinical in vitro and in vivo studies aimed at establishing the potential transfer of this cell vaccine in the clinical setting. To this end, PBMC collected from healthy donors were ex vivo activated with IL-2 and anti-CD3 antibody and in vitro treated with DHA. Results from molecular, flow cytometry and ELISA assays demonstrated that DHA treatment induced/up-regulated the expression of multiple CTAs, up-regulated the production of pro-inflammatory cytokines, but it did not alter the frequency of different immune cell populations as compared to untreated activated PBMC. Moreover, no colony growth was observed in all tested vaccine samples by the soft agar assay. Consistently, no growth of tumor mass was detected in athymic nude mice subcutaneously injected with vaccine cells. Finally, a biodistribution assay in nude mice, allowed us to identify a preferential localization of cell vaccine in the main immune cells producing organs (lymph nodes and spleen). Altogether, these results bear high translational relevance strongly supporting the potential use of this multivalent cell autologous vaccine for the treatment of malignancies of different histotype that constitutively express one or more TAAs.
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