We found that PD-1 blockade with low-dose CPM, given in combination with vaccine, synergistically induces strong antigen-specific immune responses and increases CD8+ and CD4+Foxp3− T-cell infiltration into the tumor, leading to a potent antitumor effect. Interestingly, we demonstrated that the efficacy of the combination

relies not only on CD8+ but also on CD4+ T cells. Furthermore, we found that the addition of CT-011 can enhance and prolong the effect of CPM-induced Treg-cell inhibition, simultaneously decreasing the levels of both tumor-infiltrated and splenic Treg cells. Thus, we showed for the first time that combining immune checkpoint inhibition (anti-PD-1) with Treg-cell ablation (low-dose CPM) in PD0332991 concentration the setting of vaccine is a unique strategy that leads to an effective and clinically translatable approach for the treatment of established cancer. In order to evaluate the antitumor efficacy of peptide vaccine in combination with

anti-PD-1 treatment and Treg-cell LY2835219 depletion with CPM, we used the TC-1 s.c. tumor model expressing HPV16 E7 antigen. We implanted a high number of tumor cells and chose a delayed treatment schedule to minimize the effect of vaccine and have more stringent conditions to test our treatment regimen. Mice were implanted with 50 000 TC-1 tumor cells at day 0, and by day 7 established measurable tumors (∼3-4 mm in diameter) were treated with a single low dose of CPM or PBS followed by HPV16

E7 peptide vaccine or PBS in combination with CT-011 or IgG the next day. Two more doses of vaccine and CT-011 were given on days 15 and 22 after tumor implantation (Fig. 1A). Vaccine, CT-011 or CPM alone, as well as vaccine/CT-011, vaccine/CPM or CT-011/CPM treatments resulted in different levels of tumor growth inhibition, but none led to complete regression of tumors (Fig. 1B). On day 21 after tumor implantation, the last day when all mice from all groups were still alive, Glutathione peroxidase tumor volumes of mice treated with CT-011, E7 or CPM alone were smaller compared with non-treated mice (p<0.05, p<0.001 and p<0.001, respectively) (Fig. 1C). Notably, mice that received CPM, either alone or in combination with vaccine or CT-011, had smaller tumors and prolonged survival compared with other groups, but only the combination of anti-PD-1 antibody with CPM and vaccine resulted in complete tumor regression in 50% of mice and prolonged survival compared to all other treatments (Fig. 1B and D). These experiments demonstrate that targeting PD-1, combined with a single low dose of CPM, enhances vaccine effect and allows the eradication of tumors even under stringent conditions.