The immune checkpoint blockade (ICB) immunotherapy of cancer has prolonged overall survival for some patients; however, the response rates are still low. Rational design of combination therapy targeting multiple immune checkpoint inhibitors promises to significantly improve the current ICB therapy. TIM-3 and LAG3 are highly expressed in the tumoral-infiltrating lymphocytes (TIL) and monoclonal antibodies targeting these immune inhibitors have been subjected to intensive clinical development. However, the underlying mechanisms are not fully understood. In order to dissect the mechanisms, we first systematically examined Tim-3 expression in immune cells in mouse tumors. We found Tim-3 was expressed in large portions of Treg, CD4, CD8 T cells, DC1, M1, and MDSC. Administration of a none-depleting Tim-3 antibody alone did not affect tumor progress, but combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor efficacy. Mechanistically, treatment with either Tim-3 mAbs or PD-1/Tim-3 mAbs led to a decrease of Treg in the tumor microenvironment (TME). In addition, Tim-3 mAbs and PD-1 mAbs either alone or synergistically increased IFN-γ and granzymes production by CD4+ and CD8+ TIL. Interestingly, combined treatment with Tim-3 and PD-1 mAbs greatly increased the expression of Lag3 in TIL, suggesting induction of hyperactivated effector T cells is associated with increased level of checkpoint inhibitory molecules. We further found that the antitumor efficacy with triple combination of PD-1, Tim-3, and Lag3 mAbs was much greater than double combination. Our data demonstrated that checkpoint inhibitors are genetically encoded to control the graded activation of effector T cells and simultaneous blockade of several inhibitors is necessary to fully unleash the power of immunotherapy of cancer.

Citation Format: Binfeng Lu, Min Yang, Wenwen Du, Wensi Zhai, Runzi Sun, Cuihua Yue, Jingting Jiang. Immune inhibitory receptors restrain hyperactivated effector T cells in the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B06.