Molecular characterization of an anti-cancer demethylated cellular vaccine: coding and non-coding sequencing

Therapeutic anticancer vaccines differ from preventive vaccines as they do not target factors associated with increased cancer risk; rather, they are designed to induce immunization against selected tumor antigens, thereby promoting recognition and elimination of malignant cells by the patient’s immune system. In recent years, due to the strong biological rationale and encouraging preclinical results, numerous clinical studies have focused on the application of these strategies for cancer treatment. However, to date, only two therapeutic anticancer vaccines have received approval from regulatory authorities. Among the main factors limiting their efficacy are the restricted number of antigens that can be incorporated into a single vaccine formulation and their limited immunogenicity. Furthermore, the high costs and complexity of manufacturing processes hinder access to these treatments, thereby reducing the number of patients who may benefit from them. To overcome these limitations, an innovative vaccine strategy (VAX) has been developed, based on the use of peripheral blood mononuclear cells (PBMCs) activated in vitro and subsequently treated with the epigenetic agent, decitabine, in order to modify their antigenic and immunological profile, enabling them to present antigens and induce an immune response. The present study aimed to characterize both the coding and non-coding transcriptome of multiple VAX preparations generated from healthy donor cells, in comparison with baseline PBMCs (T0) and with activated but untreated cells (untreated). To this end, total RNA extracted from each VAX, T0, and untreated preparation was analyzed using the Nanostring nCounter Sprint Profiler platform and RNA sequencing (Illumina NovaSeq 6000 System). The immunogenic potential of VAX was further evaluated at a functional level through ELISA assays for the detection of IgG antibodies specific for the tumor antigen New York esophageal squamous cell carcinoma (NY-ESO-1) in sera collected from BALB/c mice previously immunized with VAX. The results indicate that VAX displays a gene expression profile characterized by the expression of multiple tumor-associated antigens (TAA), genes involved in antigen presentation pathways, costimulatory molecules, and pro-inflammatory cytokines induced during the preparation process. In addition, epigenetic remodeling of VAX led to the induction and/or upregulation of numerous transposable elements, which predominantly influence biological processes related to immune cell proliferation and activation, while also modulating pathways associated with cell adhesion, cytokine production, and response to interferon-γ (IFN-γ). These molecular findings are functionally supported by the presence of anti–NY-ESO-1 IgG antibodies in sera from VAX-immunized mice. Overall, these data suggest that VAX possesses the capacity to elicit or enhance a specific immune response against a broad repertoire of tumor antigens, potentially resulting in therapeutic efficacy across multiple tumor histotypes and providing a solid rationale for future clinical translation.