Future cancer immunotherapies will combine multiple treatments to improve immune responses to cancer through synergistic, multi-modal mechanisms. In Phase 1 and 1b clinical trials, we found that metronomic cyclophosphamide (mCPA; 50 mg BID) enhanced the immunogenicity of a DepoVaxTM (DPX) based cancer vaccine (DPX-Survivac) in ovarian cancer patients. We reproduced these results in preclinical transplantable tumor models which allowed us to study the underlying mechanisms of cyclophosphamide-induced immune modulation, as well as explore additional combinations to enhance the therapeutic effect. In syngeneic murine models, mCPA on alternating weeks (20 mg/kg/day PO) in combination with DPX peptide vaccines provided long-term control of established tumors. Using a HPV-expressing tumor model (C3), we found that the combination of mCPA with vaccination caused selective enrichment of antigen-specific CD8+ T cells resulting in increased immune responses detected by IFN-γ ELISPOT and in vivo cytotoxicity assay, as well as improved protection from tumors. Efficacy of the vaccine and mCPA combination was limited in mice bearing advanced tumors. However, antigen-specific CD8+ T cells could be detected infiltrating the tumor by flow cytometry along with increased expression of PD-1 by RT-qPCR, suggesting that the combination therapy was able to generate strong cytotoxic T cell response but was still subject to tumor induced suppression within the tumor microenvironment. Tumor bearing mice treated with vaccine, mCPA and PD-1 blockade (with anti-PD1 or anti-PDL1) could control and induce regression of established tumors which could not be successfully treated with antibody monotherapy. Ex vivo analysis of tumor infiltrating cells demonstrated that anti-PD1 treatment did not further enhance tumor infiltration with antigen-specific CD8 T cells induced by the vaccine/ mCPA treatment, but did increase the expression of cytotoxic genes such as IFN-γ and granzyme B. This indicates that PD1 blockade may promote increased activity of activated T cells within the tumor microenvironment. We evaluated whether PD-1 blockade could enhance anti-tumor immune responses through epitope spreading by developing a mixed tumor model whereby mice were implanted with a mixture of two different tumor cell lines (C3 and B16-F10) and vaccinated with a C3-specific antigen in combination with mCPA and/or anti-PD1. Splenocytes of mice treated with the tri-therapy produced IFN-γ ELISPOT responses towards both cell lines. This effect could not be detected in tumor bearing mice treated with mCPA/anti-PD1 without vaccine. To confirm these results, we evaluated clonality of tumor infiltrating CD8 T cells using TCRβ sequencing. We conclude that enhanced tumor control mediated by the tri-therapy is mediated by highly active, tumor-specific cytotoxic T lymphocytes produced by a strongly immunogenic vaccine, resulting in epitope spreading that can further facilitate tumor rejection or control. These results provide a rationale for clinical testing of checkpoint blockade therapy in combination with our highly immunogenic combination of mCPA and DPX-Survivac.
Citation Format: Genevieve M. Weir, Olga Hrytsenko, Marianne Stanford, Mohan Karkada, Neil Berinstein, Marc Mansour. Multi-modal treatment with peptide vaccine, metronomic cyclophosphamide and PD-1 blockade provides effective tumor control through a mechanism of epitope spreading. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A137.
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