A multi-modal approach for cancer treatment: pre-clinical study of anti-tumoral and immunomodulatory efficacy of a demethylated cell-based vaccine combined with α-CTLA4 and decitabine

The overall immunosuppressive condition within the tumor context, coupled with the key contribution of DNA methylation disruption during cancer onset and development, led to synergistic combinations of immune checkpoint blockade (ICB)-based therapies with DNA hypomethylating agents (DHAs), in a multitude of cancer histotypes. Nevertheless, full responsiveness to this therapeutic approach has not been achieved. Currently, numerous clinical trials are exploiting the ability of cancer vaccines to mount a specific cancer-directed immune response as a combination strategy to ICB or epigenetic drugs. On these premises, the current study explored the therapeutic potential of integrating an innovative autologous demethylated cell-based cancer vaccine (VaxDAC) within the α-CTLA4-ICB plus decitabine (DAC)-DHA combination, in a murine tumor model engrafted with the weakly immunogenic mammary carcinoma TS/A cell line. VaxDAC consisted of (IL-2+anti-CD3ε)-activated and DAC-treated healthy murine splenocytes, with the goal to favor lymphocyte proliferation and induce an antigen-presenting cell (APC)-like phenotype, respectively. The VaxDAC-induced APC-like phenotype of immunizing cells was validated by assessing the induction of methylation-controlled cancer/testis antigens (CTAs), along with the upregulation of antigen-presenting machinery (APM) and IFN-related genes, by quantitative Real-Time PCR (qRT PCR) analyses. Additionally, immunofluorescence surface staining revealed increasing levels of MHC class I and co-stimulatory molecules, in DAC-treated splenocytes, further sustaining the use of DAC for the vaccine generation. The employment of the multi-modal combination of VaxDAC, α CTLA4, and DAC was studied in the in vivo system to assess its anti-tumor efficacy and ex vivo to evaluate its immunomodulatory potential. Results from monitoring of tumor mass growth identified in the α-CTLA4 plus DAC combinations with or without VaxDAC the two most effective in inhibiting the tumor growth, with almost overlapping trends. The immune responses elicited by VaxDAC were then investigated by ELISA and IFN-γ ELISPOT assay to monitor anti-CTA antibody production in mouse sera and CTA-specific CD8+ T cell responses from mouse lymphocytes, respectively. Data from the immune-related assays displayed the ability of VaxDAC, alone or combined, to induce the greatest production of CTA-directed antibodies, and the necessity for VaxDAC to be combined to α-CTLA4 and DAC to evoke a cellular-mediated response. Collectively, our results identified the triple combination of VaxDAC+α-CTLA4+DAC as the best therapeutic option to exert both anti-tumor and immunologic responses, compared to each monotherapy. Furthermore, the epigenetic remodeling of mouse tumors induced by systemic DAC was assessed through CTA induction and upregulation of APM and IFN-related genes, by qRT-PCR, showing gained tumor immunogenicity. Of note, the matching CTA expression between VaxDAC splenocytes and DAC-receiving tumors suggests a favored antigen recognition by the host’s immune system and further reinforces the rationale of this combination. Comprehensively, our preclinical data set the scientific rationale to translate into the clinic a multi-modal combination that employs epigenetic based therapies (i.e., innovative demethylated lymphocyte-based cancer vaccine and systemic DAC) with ICB-based immunotherapy, in the anti-tumor setting.