title: Radiolabeled humanized anti-CD3 monoclonal antibody visilizumab for imaging human T-lymphocytes.
abstract: Visilizumab is an IgG(2) humanized monoclonal antibody (mAb) characterized by non-FcgammaR binding and specific to the CD3 antigen, expressed on more than 95% of circulating resting T-lymphocytes and on activated T-lymphocytes homing in inflamed tissues. We hypothesized that the use of a radiolabeled anti-CD3 antibody might serve as a diagnostic tool for imaging T-cell traffic and lymphocytic infiltration of tissues and organs affected by autoimmune diseases. Here we describe the results of in vitro and animal experiments with (99m)Tc-succinimidyl-6-hydrazinonicotinate hydrochloride (SHNH)-visilizumab.
  
  
  METHODS
  For mAb labeling, we used a 2-step method with a heterobifunctional linker SHNH. Several titrations were performed to obtain the best labeling efficiency. In vitro quality controls included stability assay, cysteine challenge, sodium dodecyl sulfate polyacrylamide gel electrophoresis, binding assay, and immunoreactivity assay. In vivo studies by high-resolution images were performed at 6 and 24 h after the injection of (99m)Tc-SHNH-visilizumab. These included cell-targeting experiments in BALB/c mice xenografted subcutaneously with an increasing number of HuT78 cells in the leg and displaced with an excess of cold antibody. We also studied irradiated severe combined immunodeficient (SCID) mice reconstituted with human peripheral blood mononuclear cells (hPBMCs) and injected with (99m)Tc-labeled visilizumab or control mAb. After dynamic imaging for 3 h, major organs were removed, counted, and processed for immunohistologic examination.
  
  
  RESULTS
  Visilizumab was labeled with HYNIC with high labeling efficiency (>90%) and high specific activity (SA; 10,360-11,100 MBq/mg), with retained biochemical integrity and in vitro binding activity to CD3-positive cells. The in vivo targeting experiment showed a proportional increase of specific uptake with the number of injected cells, both at 6 and at 24 h, and the in vivo competition study demonstrated more than 60% decreased uptake after an excess of unlabeled antibody. In SCID mice, hPBMCs in different tissues were detected by (99m)Tc-labeled visilizumab and confirmed by histology.
  
  
  CONCLUSIONS
  Visilizumab can be efficiently labeled with (99m)Tc with high efficiency and SA and could be a valuable tool for the study of human T-lymphocyte trafficking and lymphocytic infiltration of tissues and organs.
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Malviya
authors.forename: Calogero
authors.initials: C
authors.lastname: D'Alessandria
authors.forename: Elena
authors.initials: E
authors.lastname: Bonanno
authors.forename: Vladimir
authors.initials: V
authors.lastname: Vexler
authors.forename: Roberto
authors.initials: R
authors.lastname: Massari
authors.forename: Carlo
authors.initials: C
authors.lastname: Trotta
authors.forename: Francesco
authors.initials: F
authors.lastname: Scopinaro
authors.forename: Rudi
authors.initials: R
authors.lastname: Dierckx
authors.forename: Alberto
authors.initials: A
authors.lastname: Signore
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology* / Q000493:pharmacokinetics; D061067:Antibodies, Monoclonal, Humanized; D001667:Binding, Competitive; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D002465:Cell Movement; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D051379:Mice; D015336:Molecular Probe Techniques; D009536:Niacinamide / Q000031:analogs & derivatives / Q000737:chemistry; D015609:Organotechnetium Compounds / Q000737:chemistry; D011786:Quality Control; D016719:Radioimmunodetection; D013379:Substrate Specificity; D013388:Succinimides / Q000737:chemistry; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D017252:CD3 Complex; D015609:Organotechnetium Compounds; D013388:Succinimides; C073102:succinimidyl 6-hydrazinonicotinate; D009536:Niacinamide; C456519:visilizumab