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

J Immunother Cancer. 2016 Oct 18;4:68. doi: 10.1186/s40425-016-0169-2. eCollection 2016.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Administration, Metronomic
  • Animals
  • Antineoplastic Agents, Immunological / pharmacology*
  • Antineoplastic Agents, Immunological / therapeutic use
  • B7-H1 Antigen / genetics
  • B7-H1 Antigen / metabolism
  • CD8-Positive T-Lymphocytes / drug effects
  • CD8-Positive T-Lymphocytes / immunology
  • CD8-Positive T-Lymphocytes / metabolism
  • Cancer Vaccines / immunology*
  • Cell Line, Tumor
  • Clonal Evolution / drug effects
  • Clonal Evolution / immunology
  • Cyclophosphamide / administration & dosage*
  • Cytotoxicity, Immunologic
  • Disease Models, Animal
  • Epitopes, T-Lymphocyte / chemistry
  • Epitopes, T-Lymphocyte / immunology
  • Female
  • Gene Expression
  • Humans
  • Immunomodulation / drug effects
  • Lymphocytes, Tumor-Infiltrating / drug effects*
  • Lymphocytes, Tumor-Infiltrating / immunology*
  • Lymphocytes, Tumor-Infiltrating / metabolism
  • Mice
  • Neoplasms / immunology*
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Neoplasms / therapy
  • Programmed Cell Death 1 Receptor / antagonists & inhibitors*
  • Programmed Cell Death 1 Receptor / genetics
  • Programmed Cell Death 1 Receptor / metabolism
  • T-Cell Antigen Receptor Specificity / drug effects
  • T-Cell Antigen Receptor Specificity / immunology
  • T-Lymphocyte Subsets / drug effects*
  • T-Lymphocyte Subsets / immunology*
  • T-Lymphocyte Subsets / metabolism
  • Tumor Microenvironment / drug effects
  • Tumor Microenvironment / immunology


  • Antineoplastic Agents, Immunological
  • B7-H1 Antigen
  • Cancer Vaccines
  • Epitopes, T-Lymphocyte
  • Programmed Cell Death 1 Receptor
  • Cyclophosphamide