title: Synthesis and chemoselective intramolecular crosslinking of a HER2-binding affibody.
abstract: The human epidermal growth factor receptor HER2 has emerged as an important target for molecular imaging of breast cancer. This article presents the design and synthesis of a HER2-targeting affibody molecule with improved stability and tumor targeting capacity, and with potential use as an imaging agent. The 58 aa three-helix bundle protein was assembled using solid-phase peptide synthesis, and a chemoselective ligation strategy was used to establish an intramolecular thioether bond between the side chain thiol group of a cysteine residue, positioned in the loop between helices I and II, and a chloroacetyl group on the side chain amino group of the C-terminal lysine residue. The tethered protein offered an increased thermal stability, with a melting temperature of 64 degrees C, compared to 54 degrees C for the linear control. The ligation did not have a major influence on the HER2 binding affinity, which was 320 and 380 pM for the crosslinked and linear molecules, respectively. Biodistribution studies were performed both in normal and tumor-bearing mice to evaluate the impact of the crosslinking on the in vivo behavior and on the tumor targeting performance. The distribution pattern was characterized by a low uptake in all organs except kidney, and rapid clearance from blood and normal tissue. Crosslinking of the protein resulted in a significantly increased tumor accumulation, rendering the tethered HER2-binding affibody molecule a valuable lead in the development of superior HER2 imaging agents.
authors.affiliation: School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: Torun
authors.initials: T
authors.lastname: Ekblad
authors.forename: Vladimir
authors.initials: V
authors.lastname: Tolmachev
authors.forename: Anna
authors.initials: A
authors.lastname: Orlova
authors.forename: Christofer
authors.initials: C
authors.lastname: Lendel
authors.forename: Lars
authors.initials: L
authors.lastname: Abrahmsén
authors.forename: Amelie Eriksson
authors.initials: AE
authors.lastname: Karlström
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000276:immunology*; D000918:Antibody Specificity; D003429:Cross Reactions / Q000276:immunology; D003432:Cross-Linking Reagents; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D055550:Protein Stability; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D013696:Temperature
chemical_list: D000906:Antibodies; D003432:Cross-Linking Reagents; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases