Construction and preclinical characterization of Fc-mGITRL for the immunotherapy of cancer

Purpose: To provide proper costimulation required for effective cancerT-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols. Experimental Design: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor-related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models. Results: In vivo, construct #178-14 (-5aa, -linker) showed the best activity (> 90% tumor reduction) at doses ranging from 5 to 25 lug and was found to be intact by gel electrophoresis. Similar doses used with construct #175-2 (-linker) produced good but not as high tumor regression. Construct #5-1 (+linker), which was found to be relatively unstable by SDS gel electrophoresis, produced < 60% tumor regression and required a higher dose (100 jig) to produce optimal results. Survival curves showed that Fc-mGITRL treatment extended the life of 80% of tumor-bearing mice to > 3 months compared with controls that died by day 40. T-cell depletion studies showed that CD8(+) T cells play a major role in Fc-mGITRL immunotherapy, and tumors removed from Fc-mGITRL- and DTA-1-treated mice showed a significant influx of granzyme B+ lymphocytes compared with controls. Finally,Tregulatory (Treg) cell assays showed that, unlike other Fc fusion proteins, all three Fc-mGITRL constructs profoundly suppressed Treg activity. Conclusions: These studies suggest that a stable, intact Fc-mGITRL fusion protein can provide missing costimulation for the immunotherapy of solid tumors. In addition, Fc-mGITRL may alter Treg activity to enhance its effectiveness for tumor immunotherapy.