Enhanced Cytotoxic CD8 T Cell Priming Using Dendritic Cell-Expressing Human Papillomavirus-16 E6/E7-p16INK4 Fusion Protein with Sequenced Anti-Programmed Death-1

Abstract

The incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma has increased in recent decades, though HPV prevention vaccines may reduce this rise in the future. HPV-related cancers express the viral oncoproteins E6 and E7. The latter inactivates the tumor suppressor protein retinoblastoma (Rb), which leads to the overexpression of p16(INK4) protein, providing unique Ags for therapeutic HPV-specific cancer vaccination. We developed potential adenoviral vaccines that express a fusion protein of HPV-16 E6 and E7 (Ad.E6E7) alone or fused with p16 (Ad.E6E7p16) and also encoding an anti-programmed death (PD)-1 Ab. Human monocyte-derived dendritic cells (DC) transduced with Ad.E6E7 or Ad.E6E7p16 with or without Ad.αPD1 were used to activate autologous CD8 CTL in vitro. CTL responses were tested against naturally HPV-infected head and neck squamous cell carcinoma cells using IFN-γ ELISPOT and [(51)Cr]release assay. Surprisingly, stimulation and antitumor activity of CTL were increased after incubation with Ad.E6E7p16-transduced DC (DC.E6E7p16) compared with Ad.E6E7 (DC.E6E7), a result that may be due to an effect of p16 on cyclin-dependent kinase 4 levels and IL-12 secretion by DC. Moreover, the beneficial effect was most prominent when anti-PD-1 was introduced during the second round of stimulation (after initial priming). These data suggest that careful sequencing of Ad.E6E7.p16 with Ad.αPD1 could improve antitumor immunity against HPV-related tumors and that p16 may enhance the immunogenicity of DC, through cyclin-dependent pathways, Th1 cytokine secretion, and by adding a nonviral Ag highly overexpressed in HPV-induced cancers.