Imaging of HER2-expressing tumours using a synthetic Affibody molecule containing the 99mTc-chelating mercaptoacetyl-glycyl-glycyl-glycyl (MAG3) sequence

Abstract

Purpose: 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.

Conclusion: 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.