Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging

Purpose: The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling both recombinant and synthetic production and site-specific labelling with ^99mTc or trivalent radiometals. Methods: The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow ^99mTc labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium labelling. The resulting ^99mTc-and ¹¹¹In-labelled Affibody molecules were evaluated both in vitro and in vivo. Results: Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour to blood ratio in LS174T xenografts was 30 at 4 h post-injection for both conjugates. Biodistribution data showed that the ^99mTc-labelled Affibody molecule had a fourfold lower kidney accumulation compared with the ¹¹¹In-labelled Affibody molecule while the accumulation in other organs was similar. Gamma camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Conclusion: Incorporation of the C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt or yttrium) and for a flexible production (chemical synthesis or recombinant).