Tumor microenvironment (TME) is characterized by multiple immune suppressive mechanisms able to suppress anti-tumor effector cell immunity. Combinatorial strategies, including vaccine and immunomodulatory drugs, need to be developed for improved immunotherapy efficacy. A novel combinatorial approach was assessed in C57BL/6 mice injected with mouse melanoma B16F10 cells. A multi-peptide vaccine (PEPT) was combined with a low dose metronomic chemotherapy (MCT) and an anti-PD-1 checkpoint inhibitor (CI). Statistical analysis were performed with the unpaired two-sided Student's t-test and ANOVA. Animals treated with the multi-peptide vaccine combined with MCT or CI showed remarkable delay in tumor growth and prolonged survival as compared to control groups. The multi-pronged combination including PEPT+MCT+CI was able to prolong survival in all mice and inhibit tumor growth in 66.6% of mice. All animals which did not show tumor growth were re-challenged with the same melanoma cells and one of them showed complete tumor growth inhibition. The anti-tumor effect was associated with strong T cell immune response to vaccine mutated peptides and significant reduction of regulatory T cells. The combination of a vaccine with MCT and CI was highly efficient in potentiating the vaccine's anti-tumor effects. The approach is highly promising to be moved into clinical trial.Keywords:anti PD-1; cancer vaccine; combinatorial strategy; metronomic chemotherapy; mutated epitopes.

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