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J Immunother Cancer. 2016 Oct 18;4:68. eCollection 2016.

Anti-PD-1 increases the clonality and activity of tumor infiltrating antigen specific T cells induced by a potent immune therapy consisting of vaccine and metronomic cyclophosphamide.

Author information

1
Immunovaccine Inc., 1344 Summer St., Halifax, NS B3H 0A8 Canada.
2
Sunnybrook Research Institute, 2075 Bayview Ave., Toronto, ON M4N 3M5 Canada ; University of Toronto, 27 King's College Cir, Toronto, ON M5S 1A1 Canada.
3
Immunovaccine Inc., 1344 Summer St., Halifax, NS B3H 0A8 Canada ; Department of Microbiology & Immunology, Dalhousie University, 5850 College St., Room 7C, Halifax, NS B3H 4R2 Canada.

Abstract

BACKGROUND:

Future cancer immunotherapies will combine multiple treatments to generate functional immune responses to cancer antigens through synergistic, multi-modal mechanisms. In this study we explored the combination of three distinct immunotherapies: a class I restricted peptide-based cancer vaccine, metronomic cyclophosphamide (mCPA) and anti-PD-1 treatment in a murine tumor model expressing HPV16 E7 (C3).

METHODS:

Mice were implanted with C3 tumors subcutaneously. Tumor bearing mice were treated with mCPA (20 mg/kg/day PO) for seven continuous days on alternating weeks, vaccinated with HPV16 E749-57 peptide antigen formulated in the DepoVax (DPX) adjuvanting platform every second week, and administered anti-PD-1 (200 μg/dose IP) after each vaccination. Efficacy was measured by following tumor growth and survival. Immunogenicity was measured by IFN-γ ELISpot of spleen, vaccine draining lymph nodes and tumor draining lymph nodes. Tumor infiltration was measured by flow cytometry for CD8α+ peptide-specific T cells and RT-qPCR for cytotoxic proteins. The clonality of tumor infiltrating T cells was measured by TCRβ sequencing using genomic DNA.

RESULTS:

Untreated C3 tumors had low expression of PD-L1 in vivo and anti-PD-1 therapy alone provided no protection from tumor growth. Treatment with DPX/mCPA could delay tumor growth, and tri-therapy with DPX/mCPA/anti-PD-1 provided long-term control of tumors. We found that treatment with DPX/mCPA/anti-PD-1 enhanced systemic antigen-specific immune responses detected in the spleen as determined by IFN-γ ELISpot compared to those in the DPX/mCPA group, but immune responses in tumor-draining lymph nodes were not increased. Although no increases in antigen-specific CD8α+ TILs could be detected, there was a trend for increased expression of cytotoxic genes within the tumor microenvironment as well as an increase in clonality in mice treated with DPX/mCPA/anti-PD-1 compared to those with anti-PD-1 alone or DPX/mCPA. Using a library of antigen-specific CD8α+ T cell clones, we found that antigen-specific clones were more frequently expanded in the DPX/mCPA/anti-PD-1 treated group.

CONCLUSIONS:

These results demonstrate how the efficacy of anti-PD-1 may be improved by combination with a potent and targeted T cell activating immune therapy.

KEYWORDS:

Clonality; Metronomic cyclophosphamide; PD-1; Tumor microenvironment; Vaccine

PMID:
27777777
PMCID:
PMC5067905
DOI:
10.1186/s40425-016-0169-2
[Indexed for MEDLINE]
Free PMC Article

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