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Construction and Preclinical Characterization of Fc-mGITRL for the Immunotherapy of Cancer

Authors' Affiliations: Departments of ¹ Pathology and ² Microbiology, Keck School of Medicine at the University of Southern California, Los Angeles, California and ³ Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland

Requests for reprints: Alan L. Epstein, Department of Pathology, Keck School of Medicine, University of Southern California, 2011 Zonal Avenue, HMR 205, Los Angeles, CA 90033. Phone: 323-442-1172; Fax: 323-342-3049; E-mail: aepstein{at}usc.edu.

Purpose: To provide proper costimulation required for effective cancer T-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 µg 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 µg) 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, T regulatory (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.