title: Construction and preclinical characterization of Fc-mGITRL for the immunotherapy of cancer.
abstract: 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.
authors.affiliation: Department of Pathology, Keck School of Medicine at the University of Southern California, Los Angeles, CA 90033, USA.
authors.forename: Peisheng
authors.initials: P
authors.lastname: Hu
authors.forename: Robyn S
authors.initials: RS
authors.lastname: Arias
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Sadun
authors.forename: Yu-Chih
authors.initials: YC
authors.lastname: Nien
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.forename: Helen
authors.initials: H
authors.lastname: Sabzevari
authors.forename: M E Christine
authors.initials: ME
authors.lastname: Lutsiak
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Khawli
authors.forename: Alan L
authors.initials: AL
authors.lastname: Epstein
mesh_terms: 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*
chemical_list: C508594:IL2 protein, human; D007141:Immunoglobulin Fc Fragments; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; C478966:Tnfsf18 protein, mouse; D048069:Tumor Necrosis Factors