Abstract B91: Anti-PD1 blockade increases the activity of tumor infiltrating antigen-specific CD8 T cells induced by a potent peptide vaccine in combination with metronomic cyclophosphamide
Authors: Weir Genevieve , Hrytsenko Olga , Stanford Marianne , Karkada Mohan , Berinstein Neil , Mansour Marc 
Published: May 2016

Abstract
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 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 anti-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, however, RT-qPCR analysis of the tumor demonstrated an increase in expression of cytotoxic T cell gene signatures with anti-PD1 treatment within the tumor; these results were confirmed in RT-qPCR analysis of tumor draining lymph node. An increased expression of PDL-1 was also detected in all vaccinated mice, regardless of whether they were also treated with anti-PD1. 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. 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 Weir, Olga Hrytsenko, Marianne Stanford, Mohan Karkada, Neil Berinstein, Marc Mansour. Anti-PD1 blockade increases the activity of tumor infiltrating antigen-specific CD8 T cells induced by a potent peptide vaccine in combination with metronomic cyclophosphamide. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B91.