Abstract
Purpose: To generate and characterize a murine GITR ligand fusion protein (mGITRL-FP) designed to maximize valency and the potential to agonize the GITR receptor for cancer immunotherapy.Experimental Design: The EC50 value of the mGITRL-FP was compared with an anti-GITR antibody in an in vitro agonistic cell-based reporter assay. We assessed the impact of dose, schedule, and Fc isotype on antitumor activity and T-cell modulation in the CT26 tumor model. The activity of the mGITRL-FP was compared with an agonistic murine OX40L-FP targeting OX40, in CT26 and B16F10-Luc2 tumor models. Combination of the mGITRL-FP with antibodies targeting PD-L1, PD-1, or CTLA-4 was analyzed in mice bearing CT26 tumors.Results: The mGITRL-FP had an almost 50-fold higher EC50 value compared with an anti-murine GITR antibody. Treatment of CT26 tumor-bearing mice with mGITRL-FP-mediated significant antitumor activity that was dependent on isotype, dose, and duration of exposure. The antitumor activity could be correlated with the increased proliferation of peripheral CD8+ and CD4+ T cells and a significant decrease in the frequency of intratumoral Tregs. The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment.Conclusions: These results suggest that therapeutically targeting GITR represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody. In addition, these data provide, for the first time, early proof of concept for the potential combination of GITR targeting agents with OX40 agonists and PD-L1 antagonists. Clin Cancer Res; 23(13); 3416-27. ©2017 AACR.
Highlights
The function of T cells is tightly regulated to maintain peripheral tolerance and ensure their safe and effective activation
The combination of mGITRL-FP with mOX40L-FP or checkpoint inhibitor antagonists enhanced antitumor immunity above that of monotherapy treatment. These results suggest that therapeutically targeting Glucocorticoid-induced TNFR-related protein (GITR) represents a unique approach to cancer immunotherapy and suggests that a multimeric fusion protein may provide increased agonistic potential versus an antibody
When the purified denatured mGITRL-FP was visualized by SDS-PAGE (Fig. 1B), it demonstrated a high degree of homogeneity and a molecular weight that was slightly higher than the expected molecular weight of 48 kDa presumably due to glycosylation of the fragment crystallizable (Fc) and mGITRL domains
Summary
The function of T cells is tightly regulated to maintain peripheral tolerance and ensure their safe and effective activation. One critical mechanism of control is a series of cell surface receptors, expressed on T cells themselves, which deliver inhibitory or stimulatory cosignals in a spatially and temporally controlled manner to control T-cell activation in response to antigen. The interaction between GITR and its ligand delivers positive costimulatory signals to T cells, which enhance their proliferation and activation [2, 3] and increase their resistance to suppression by Tregs [4,5,6,7]. These signals have been demonstrated to directly modulate the function and reduce the stability of Tregs [8,9,10]
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