Significant therapeutic efficacy of blocking T cell negative regulatory pathway in vivo

4884 Recent studies have suggested that tumors might escape from host immunity through PD-L/PD-1 interaction. We have recently reported that PD-L expression in human esophageal cancer is an independent prognostic factor (Clin Cancer Res. 2005,11:2947). In the following study, we have also found that PD-L1 expression has similar prognostic value in pancreatic cancer. These clinical data suggest that PD-L/PD-1 pathway may play a pivotal role in human cancer including these aggressive and intractable cancers. In this study, we have examined therapeutic efficacy of targeting T cell negative pathway toward clinical application. To this end, we utilized murine syngeneic tumor models. In hepatic metastasis model, a murine colon cancer, CT26 was inoculated into the spleen in syngeneic BALB/c mice. We treated mice with anti-PD-1 (RMP1-14), anti-PD-L1 (MIH-5), anti-PD-L2 (TY25) mAb or control Ig starting on the day of tumor inoculation. Hepatic metastasis has been significantly inhibited by either anti-PD-1 or PD-L2, but not PD-L1 mAb compared to controls at 3 weeks after inoculation (the median number of metastases; anti-PD-1, 5, anti-PD-L1, 25, anti-PD-L2, 7, control Ig, 27). The in vivo experiments of CD4 or CD8 T cell depletion revealed that the inhibition of tumor metastasis by PD-1 blockade was mainly mediated by CD8 T cell subset. Interestingly, the coadministration of anti-CTLA-4 mAb (4F10) with PD-1 blockade further enhanced antitumor immunity and resulted in complete inhibition of liver metastasis, although CTLA-4 blockade alone was less effective and insufficient for complete inhibition. Thus, the combination of PD-1 and CTLA-4 blockade displayed synergistic effects on the inhibition of tumor metastasis. Then, we applied this combined blockade therapy of PD-1 and CTLA-4 to the advanced tumor. CT26 was subcutaneously inoculated in the flank of mice. When tumor reached 6-8 mm in diameter around 10 days after inoculation, the treatment of both anti-PD-1 and anti-CTLA-4 mAbs was started. Compared to mice treated with control Ig, the simultaneous blockade of PD-1 and CTLA-4 induced substantial antitumor effect in vivo. Tumors were either completely rejected or significantly inhibited in all mice by 2 weeks after treatment. By histology, much more cell recruitments and necrosis in the established tumor were observed during mAb treatment compared to control. Immunohistochemical analysis revealed that the treatment promoted the infiltrations of tumor-reactive T cells, predominantly CD8 T cell, into the tumor. There was no overt toxicity in mice during and after the treatment. In conclusion, we have demonstrated for the first time that simultaneous blockade of differential T cell negative regulatory pathways of PD-L/PD-1 and B7/CTLA-4 evoked powerful tumor immunity in vivo. Thus, we suggest that this promising strategy is potentially effective in treatment of tumor metastasis and advanced tumors in humans.