title: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule.
abstract: BACKGROUND
  The cellular binding and processing of an epidermal growth factor receptor (EGFR) targeting affibody molecule, (Z(EGFR:955))(2), was studied. This new and small molecule is aimed for applications in nuclear medicine. The natural ligand epidermal growth factor (EGF) and the antibody cetuximab were studied for comparison.
  
  
  METHODS
  All experiments were made with cultured A431 squamous carcinoma cells. Receptor specificity, binding time patterns, retention and preliminary receptor binding site localization studies were all made after (125)I labeling. Internalization was studied using Oregon Green 488, Alexa Fluor 488 and CypHer5E markers.
  
  
  RESULTS
  [(125)I](Z(EGFR:955))(2) and [(125)I]cetuximab gave a maximum cellular uptake of (125)I within 4 to 8 h of incubation, while [(125)I]EGF gave a maximum uptake already after 2 h. The retention studies showed that the cell-associated fraction of (125)I after 48 h of incubation was approximately 20% when delivered as [(125)I](Z(EGFR:955))(2) and approximately 25% when delivered as [(125)I]cetuximab. [(125)I]EGF-mediated delivery gave a faster (125)I release, where almost all cell-associated radioactivity had disappeared within 24 h. All three substances were internalized as demonstrated with confocal microscopy. Competitive binding studies showed that both EGF and cetuximab inhibited binding of (Z(EGFR:955))(2) and indicated that the three substances competed for an overlapping binding site.
  
  
  CONCLUSIONS
  The results gave information on cellular processing of radionuclides when delivered with (Z(EGFR:955))(2) in comparison to delivery with EGF and cetuximab. Competition assays suggested that [(125)I](Z(EGFR:955))(2) bind to Domain III of EGFR. The affibody molecule (Z(EGFR:955))(2) can be a candidate for EGFR imaging applications in nuclear medicine.
authors: Rudbeck Laboratory, Department of Oncology, Radiology and Clinical Immunology, Biomedical Radiation Sciences, Uppsala University, SE-751 85 Uppsala, Sweden. erika.nordberg@bms.uu.se
authors: Erika
authors: E
authors: Nordberg
authors: Mikaela
authors: M
authors: Friedman
authors: Lovisa
authors: L
authors: Göstring
authors: Gregory P
authors: GP
authors: Adams
authors: Hjalmar
authors: H
authors: Brismar
authors: Fredrik Y
authors: FY
authors: Nilsson
authors: Stefan
authors: S
authors: Ståhl
authors: Bengt
authors: B
authors: Glimelius
authors: Jörgen
authors: J
authors: Carlsson
mesh_terms: D000911:Antibodies, Monoclonal / Q000493:pharmacokinetics; D061067:Antibodies, Monoclonal, Humanized; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D002264:Carboxylic Acids; D045744:Cell Line, Tumor; D000068818:Cetuximab; D004815:Epidermal Growth Factor / Q000493:pharmacokinetics; D066246:ErbB Receptors / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005464:Fluorobenzenes; D006801:Humans; D007457:Iodine Radioisotopes / Q000493:pharmacokinetics; D007553:Isotope Labeling; D018613:Microscopy, Confocal; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics*
chemical_list: C513915:AlexaFluor 488 carboxylic acid tetrafluorophenyl ester; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D002264:Carboxylic Acids; D005464:Fluorobenzenes; D007457:Iodine Radioisotopes; C436864:Oregon Green 488 carboxylic acid; D019275:Radiopharmaceuticals; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D000068818:Cetuximab