Improved Anti-tumor Immunity Using Xenogenic Melanoma DNA Vaccines Combined With Adjuvant In Murine Melanoma

Malignant melanoma is a malignant tumor with highly aggressive and melanoma mortality worldwide remains high. With the development of melanoma-associated antigen including tyrosinase related protein2(TRP-2) and gp100, melanoma DNA vaccine has developed greatly in recent years. Despite gratifying achievements have been obtained in the treatment of malignant melanoma, melanoma DNA vaccines are still many issues, such aseasy to cause immune tolerance and the immune response induced weak, to be resolved.TRP-2and gp100are highly expressed in both human and mouse melanoma. Mouse gp100(mgp100) and mouse TRP-2(mTRP-2) in mice prone to cause immune tolerance,which resulted in poorly immune response. In this study, we used xenogenic antigens tobreak these immune tolerance and enhance the anti-tumor efficacy of the melanoma DNA vaccine. DNA vaccines, however, are not considered as strong immunogens and the use of appropriate adjuvant are required to improve its effect. We thus chose Ii-PADRE(invariant Pan DR reactive epitope) and murine interleukin-12(mIL-12) combined with ourmelanoma DNA vaccine. Furthermore, regulatory T (Treg) cells in cancer patients may inhibit the anti-tumor immune response against self-tumor antigen like TRP-2and gp100.To make the vaccine more effective, we used ONTAK (denileukin diftitox) removal of Tregs.Our results indicated that immunization with melanoma DNA vaccines (phgp100and phTRP-2) encoding human gp100(hgp100) and human TRP-2(hTRP-2) showed significant protection in a B16F10challenge model. Therefore, xenogenic gp100and TRP-2appears necessary in breaking tolerance for these antigens. Co-administration of phgp100, phTRP-2and pIi-PADRE increased two-fold the number of IFN-γ secreting cells against TRP-2in the ELISPOT assay, and increased four-fold the in vitro cytotoxicity against B16F10cells. While combination of phTRP-2, phgp100and pIi-PADRE (immunization intramusc ularly) with3times injection of pmIL-12(immunization intratumorally) showed the besttherapeutic effect, in which the tumor-free survival was75%and some mice showed thegreatest tumor regression of establish B16F10tumors. Moreover, we have shown that asingle intraperitoneal injection of ONTAK one day before phTRP-2and phgp100vaccination was sufficient to enhance the anti-tumor effect.In conclusion, co-immunization of two xenogenic melanoma DNA vaccine (phgp100and phTRP-2) can significantly improve the protective effect against B16F10melanoma; depletion of Tregs by ONTAK enhancement of the cancer vaccine efficacy; while the optimal strategy is co-immunization of phgp10, phTRP-2and gene adjuvant pIi-PADRE, pmIL-12. This treatment regime resulted in complete regression of established B16F10tumors independent of Treg depletion, long-term mice survival and protection from repeated challenge.