title: 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.
abstract: 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).
  
  
  
  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 E749-57peptide 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.
  
  
  
  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.
  
  
  
  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.
authors: Immunovaccine Inc., 1344 Summer St., Halifax, NS B3H 0A8 Canada.
authors: Genevieve M
authors: GM
authors: Weir
authors: Immunovaccine Inc., 1344 Summer St., Halifax, NS B3H 0A8 Canada.
authors: Olga
authors: O
authors: Hrytsenko
authors: Immunovaccine Inc., 1344 Summer St., Halifax, NS B3H 0A8 Canada.
authors: Tara
authors: T
authors: Quinton
authors: Sunnybrook Research Institute, 2075 Bayview Ave., Toronto, ON M4N 3M5 Canada ; University of Toronto, 27 King's College Cir, Toronto, ON M5S 1A1 Canada.
authors: Neil L
authors: NL
authors: Berinstein
authors: 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.
authors: Marianne M
authors: MM
authors: Stanford
authors: Immunovaccine Inc., 1344 Summer St., Halifax, NS B3H 0A8 Canada.
authors: Marc
authors: M
authors: Mansour
mesh_terms: D059250:Administration, Metronomic; D000818:Animals; D000074322:Antineoplastic Agents, Immunological / Q000494:pharmacology* / Q000627:therapeutic use; D060890:B7-H1 Antigen / Q000235:genetics / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D019496:Cancer Vaccines / Q000276:immunology*; D045744:Cell Line, Tumor; D060965:Clonal Evolution / Q000187:drug effects / Q000276:immunology; D003520:Cyclophosphamide / Q000008:administration & dosage*; D003602:Cytotoxicity, Immunologic; D004195:Disease Models, Animal; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology; D005260:Female; D015870:Gene Expression; D006801:Humans; D056747:Immunomodulation / Q000187:drug effects; D016246:Lymphocytes, Tumor-Infiltrating / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009369:Neoplasms / Q000276:immunology* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D061026:Programmed Cell Death 1 Receptor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D037182:T-Cell Antigen Receptor Specificity / Q000187:drug effects / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D059016:Tumor Microenvironment / Q000187:drug effects / Q000276:immunology
chemical_list: D000074322:Antineoplastic Agents, Immunological; D060890:B7-H1 Antigen; D019496:Cancer Vaccines; D018984:Epitopes, T-Lymphocyte; D061026:Programmed Cell Death 1 Receptor; D003520:Cyclophosphamide
keywords: Clonality; Metronomic cyclophosphamide; PD-1; Tumor microenvironment; Vaccine