Direct comparison of ¹¹¹In-labelled two-helix and three-helix Affibody molecules for in vivo molecular imaging

Purpose Radiolabelled Affibody molecules have demonstrated a potential for visualization of tumour-associated molecular targets. Affibody molecules (7 kDa) are composed of three alpha-helices. Recently a smaller two-helix variant of Affibody molecules (5.1 kDa) was developed. The aim of this study was to compare two- and three-helix HER2-targeting Affibody molecules directly in vivo. Methods The three-helix Affibody molecule ABY-002 and the two-helix Affibodymolecule PEP09239 were labelled with 111In at the N-termini via DOTA chelator. Tumour-targeting propertieswere directly compared at 1 and 4 h after injection in mice bearing SKOV-3 xenografts with high HER2 expression and LS174T xenografts with low HER2 expression. Results The dissociation constants (KD) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. 111In-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of ¹¹¹In-ABY-002 was significantly higher than that of 111In-PEP09239. The tumour-toblood ratio was higher for 111In-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of ¹¹¹In-ABY- 002 was eightfold higher than that of 111In-PEP09239 in xenografts with low expression. Tumour-to-blood ratios were equal in this case, but other tumour-to-organ ratios were appreciably higher for the three-helix variant. Conclusion For tumours with high HER2 expression, twohelix HER2-targeting Affibody molecules can provide higher tumour-to-blood ratio at the cost of lower tumour uptake. In the case of low expression, both tumour uptake and tumourto- organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.