Construction and preclinical characterization of Fc-mGITRL for the immunotherapy of cancer.
OBJECTIVE
To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols.


METHODS
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 microg 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 microg) 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.
Hu
Peisheng
P
Department of Pathology, Keck School of Medicine at the University of Southern California, Los Angeles, CA 90033, USA.
Arias
Robyn S
RS
Sadun
Rebecca E
RE
Nien
Yu-Chih
YC
Zhang
Nan
N
Sabzevari
Helen
H
Lutsiak
M E Christine
ME
Khawli
Leslie A
LA
Epstein
Alan L
AL
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000276:immunology; D007141:Immunoglobulin Fc Fragments / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D007167:Immunotherapy; D007376:Interleukin-2 / Q000096:biosynthesis / Q000276:immunology; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism; D048069:Tumor Necrosis Factors / Q000008:administration & dosage / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*
C508594:IL2 protein, human; D007141:Immunoglobulin Fc Fragments; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; C478966:Tnfsf18 protein, mouse; D048069:Tumor Necrosis Factors