title: Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence.
abstract: OBJECTIVE
  Expression of human epidermal growth factor receptor type 2 (HER2) in malignant tumours possesses well-documented prognostic and predictive value. Non-invasive imaging of expression can provide valuable diagnostic information, thereby influencing patient management. Previously, we reported a phage display selection of a small (about 7 kDa) protein, the Affibody molecule Z(HER2:342), which binds HER2 with subnanomolar affinity, and demonstrated the feasibility of targeting of HER2-expressing xenografts using radioiodinated Z(HER2:342). The goal of this study was to develop a method for (99m)Tc labelling of Z(HER2:342) using the MAG3 chelator, which was incorporated into Z(HER2:342) using peptide synthesis, and evaluate the targeting properties of the labelled conjugate.
  
  
  METHODS
  MAG3-Z(HER2:342) was assembled using Fmoc/tBu solid phase peptide synthesis. Biochemical characterisation of the agent was performed using RP-HPLC, ESI-MS, biosensor studies and circular dichroism. A procedure for (99m)Tc labelling in the presence of sodium/potassium tartrate was established. Tumour targeting was evaluated by biodistribution study and gamma camera imaging in xenograft-bearing mice. Biodistribution of (99m)Tc-MAG3-Z(HER2:342) and (125)I-para-iodobenzoate -Z(HER2:342) was compared 6 h p.i.
  
  
  RESULTS
  Synthetic MAG3-Z(HER2:342) possessed an affinity of 0.2 nM for HER2 receptors. The peptide was labelled with (99m)Tc with an efficiency of about 75-80%. Labelled (99m)Tc-MAG3-Z(HER2:342) retained capacity to bind specifically HER2-expressing SKOV-3 cells in vitro. (99m)Tc-MAG3-Z(HER2:342) showed specific tumour targeting with a contrast similar to a radioiodinated analogue in mice bearing LS174T xenografts. Gamma camera imaging demonstrated clear and specific visualisation of HER2 expression.
  
  
  CONCLUSIONS
  Incorporation of a mercaptoacetyl-containing chelating sequence during chemical synthesis enabled site-specific (99m)Tc labelling of the Z(HER2:342) Affibody molecule with preserved targeting capacity.
authors.affiliation: School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Torun
authors.initials: T
authors.lastname: Engfeldt
authors.affiliation: Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.|Affibody AB, Bromma, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.affiliation: Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Thuy
authors.initials: T
authors.lastname: Tran
authors.affiliation: Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors.forename: Alexander
authors.initials: A
authors.lastname: Bruskin
authors.affiliation: Department of Hospital Physics, Uppsala University Hospital, Uppsala, Sweden.
authors.forename: Charles
authors.initials: C
authors.lastname: Widström
authors.affiliation: School of Biotechnology, Division of Molecular Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Amelie Eriksson
authors.initials: AE
authors.lastname: Karlström
authors.affiliation: Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden. Vladimir.Tolmachev@bms.uu.se.|Affibody AB, Bromma, Sweden. Vladimir.Tolmachev@bms.uu.se.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
mesh_terms: D000818:Animals; D000906:Antibodies; D015374:Biosensing Techniques; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000175:diagnosis* / Q000000981:diagnostic imaging*; D010455:Peptides / Q000737:chemistry; D011877:Radionuclide Imaging; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D017263:Technetium Tc 99m Mertiatide / Q000493:pharmacokinetics
chemical_list: D000906:Antibodies; D010455:Peptides; D019275:Radiopharmaceuticals; D018719:Erb-b2 Receptor Tyrosine Kinases; D017263:Technetium Tc 99m Mertiatide; C508053:ERBB2 protein, human