title: In Vivo Imaging of Fluorescent Probes Linked to Antibodies Against Human and Rat Vascular Endothelial Growth Factor.
abstract: OBJECTIVE
  Anti-VEGF therapy has improved functional outcome for many patients with neovascular AMD. A particular challenge in routine clinical application is to find the best treatment regimen as a high degree of interindividual variability of disease activity has been noted. The aim of the study was to investigate fluorescent probes linked to antibodies against VEGF for in vivo imaging in an animal model.
  
  
  METHODS
  Bevacizumab, B20-4.1.1 and AF564 were covalently attached to the novel dye 6S-indocyanine green (ICG) maleimide. Binding and proliferation properties were assessed. In a rat model of laser-induced choroidal neovascularization, retinal uptake and topographic localization of antibody-conjugates were analyzed. Distribution and accumulation of the probes were determined by immunohistochemistry and flow cytometry analysis.
  
  
  RESULTS
  Antibody-conjugates retained target binding affinity and showed no toxicity. In vivo imaging showed a strong fluorescence immediately following an intravenous or intravitreal injection. While accumulation within the laser lesions was visualized for all three antibody conjugates, the signal strength and the duration of fluorescence varied. In addition, distinct fluorescent spots were also recognized. Patterning and in-depth analyses including histology and flow cytometry data strongly suggest that the fluorescent spots represent labeled microglial cells and/or macrophages.
  
  
  CONCLUSIONS
  Pharmacokinetics of fluorescent-labeled bevacizumab, B20-4.1.1 and AF564 can be investigated in vivo. In this model, interpretation of long-term in vivo observations is difficult because of a possible rat-specific immune response and challenges to image localized binding of soluble VEGF. Further investigations in a primate model and the use of appropriate antibodies directed against the VEGF-receptor may represent alternative approaches.
authors.affiliation: Department of Ophthalmology University of Bonn, Bonn, Germany.
authors.forename: Johanna H
authors.initials: JH
authors.lastname: Meyer
authors.affiliation: Department of Ophthalmology University of Bonn, Bonn, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Cunea
authors.affiliation: mivenion GmbH, Berlin, Germany.
authors.forename: Kai
authors.initials: K
authors.lastname: Licha
authors.affiliation: mivenion GmbH, Berlin, Germany.
authors.forename: Pia
authors.initials: P
authors.lastname: Welker
authors.affiliation: Department of Ophthalmology University of Bonn, Bonn, Germany.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Sonntag-Bensch
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Wafula
authors.affiliation: Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
authors.affiliation: Institute of Biostatistics, University of Bonn, Bonn, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Fimmers
authors.affiliation: Department of Ophthalmology University of Bonn, Bonn, Germany.
authors.forename: Frank G
authors.initials: FG
authors.lastname: Holz
authors.affiliation: Department of Ophthalmology University of Bonn, Bonn, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Schmitz-Valckenberg
mesh_terms: D000818:Animals; D000068258:Bevacizumab / Q000008:administration & dosage / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D020256:Choroidal Neovascularization / Q000378:metabolism; D004195:Disease Models, Animal; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000032:analysis* / Q000493:pharmacokinetics; D006801:Humans; D018796:Immunoconjugates / Q000008:administration & dosage / Q000032:analysis* / Q000276:immunology / Q000493:pharmacokinetics; D007150:Immunohistochemistry / Q000379:methods; D007208:Indocyanine Green / Q000008:administration & dosage / Q000032:analysis* / Q000737:chemistry / Q000493:pharmacokinetics; D058449:Intravitreal Injections; D007834:Lasers; D008028:Light Coagulation / Q000379:methods; D008297:Male; D061848:Optical Imaging / Q000379:methods*; D051381:Rats; D016896:Treatment Outcome; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology*
chemical_list: D005456:Fluorescent Dyes; D018796:Immunoconjugates; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; D007208:Indocyanine Green