To counteract tumor-induced immune suppression, cancer vaccines are increasingly being combined with immune modulators that can not only reverse immune suppression but also enhance vaccine induced immune responses. We found that metronomic cyclophosphamide (50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients in a phase I clinical study. We emulated these results using transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide induced immune modulation. In several different models, mice were given mCPA on alternating weeks (20 mg/kg/day PO) in combination with a DPX vaccine containing relevant peptide antigens every three weeks. In these models, only the combination provided effective and significant long-term control of tumor growth. Notably, we found that the efficacy of the combination was comparable when vaccine was given at the beginning or end of a cycle of mCPA. Metronomic CPA had a pronounced lymphodepletive effect on the vaccine draining lymph node, yet did not reduce the development of antigen-specific CD8+ T cells induced by vaccination as detected by MHC-multimer flow cytometry. Combination treatment also increased cytotoxic T cell activity in the spleen measured by IFN-γ ELISPOT and in vivo cytotoxic T cell assay. Analysis of immune gene signatures in the tumor microenvironment by RT-qPCR detected elevated levels of cytotoxic markers, such as IFN-γ and granzyme B, as well as co-inhibitory markers, such as PD-1 and CTLA-4, in mice treated with combination therapy, the latter providing a strong rational for modulating these pathways with recently available monoclonal antibodies. Analysis of spleen cell populations by flow cytometry indicated that mCPA induced transient lymphodepletion that was marked by a selective expansion of myeloid-derived suppressor cells in the absence of vaccination. Selective depletion of regulatory T cells was not observed, in contrast to other regimens of low dose CPA. These results demonstrate that mCPA provides a complex form of immune modulation that is most effective when combined with active immunization. Using these models we can evaluate other immune modulator agents that may enhance vaccine activity, including checkpoint inhibitors or other immune based therapies.

Citation Format: Genevieve Weir, Olga Hrytsenko, Marianne M. Stanford, Neil L. Berinstein, Mohan Karkada, Robert S. Liwski, Marc Mansour. Metronomic cyclophosphamide enhances the immunogenicity and anti-tumor activity of a DepoVax based vaccine and may be further enhanced with inhibitors of CTLA-4 or PD-1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5028. doi:10.1158/1538-7445.AM2014-5028