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°C, compared to 54°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. © 2009 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 92: 116–123, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com