TRIPLE IMMUNOTHERAPY TO OVERCOME IMMUNE EVASION BY TUMOR IN A MELANOMA MOUSE MODEL

Abstract

Background: This study devises a triple immunotherapy to treat melanoma in a mouse model. The combination includes anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) antibodies, monophosphoryl-lipid-A (MPLA), and an indolamine-dioxygenase-1 (IDO1) inhibitor. The aim of the study is, first, to rule out any major toxic effects related to this therapy and, second, to assess its antitumor effects. Methods: Cancer-free C57BL/6 mice were randomized into control groups and groups receiving single, dual, or triple therapies of the defined treatments. Clinical signs, weight gain, and histological sections from their main organs were assessed. Then, melanomabearing mice were segregated into similar groups, monitored for survival, and their tumor size was measured repeatedly. Flow cytometry was used to analyze immune cell populations in the tumor masses including CD4+, CD8+, and regulatory T cells in addition to natural killer cells. Finally, serum levels of interleukin-12 (IL-12), vascular endothelial growth factor (VEGF) and S100 calcium-binding protein B (S100B) were quantified using enzyme-linked immunosorbent assay (ELISA). Results: No adverse effects were detected in any of the treated groups. Survival analysis indicated that the groups receiving dual or triple therapies had prolonged survival compared to the controls. However, the group receiving triple therapy was the only group to show statistically significant increase in survival compared to the controls. Tumor size progression paralleled the survival outcome. The group receiving the triple therapy showed statistically significant smaller tumor sizes compared to all the other groups throughout the whole monitoring period. Flow cytometry used to analyze immune cell populations in the tumor mass indicated that the triple immune therapy was capable of significantly enhancing the natural killer cell counts as well as the CD3+CD4+/Treg and CD3+CD8+/Treg ratios possibly enhancing the anti-tumorigenic environment. While serum levels of the tumorsuppressive IL-12 came opposed to the expected by being lowest in the group with the most favorable outcome, circulating VEGF and S100B levels were below detection level in the triple immunotherapy group through all detection time points and hence were in accordance with the survival and tumor progression results. Conclusion: Generated data rule out any major adverse events pertaining to the triple immunotherapy and reveal its enhanced effectiveness in thwarting tumor progression over all other tested treatments. This outcome is mainly achieved through the enhancement of natural killer cells and the ratios of CD4+ and CD8+ T-cells to regulatory T-cells.