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

purposeRadiolabelled 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.

methodsThe three-helix Affibody molecule ABY-002 and the two-helix Affibody molecule PEP09239 were labelled with (111)In at the N-termini via DOTA chelator. Tumour-targeting properties were 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.

resultsThe dissociation constants (K(D)) for HER2 binding were 78 pM for the three-helix Affibody molecule and 2.1 nM for the two-helix Affibody molecule. (111)In-PEP09239 cleared more rapidly from the blood. In xenografts with high HER2 expression, the uptake of (111)In-ABY-002 was significantly higher than that of (111)In-PEP09239. The tumour-to-blood ratio was higher for (111)In-PEP09239 at 4 h after injection, while there was no significant difference in other tumour-to-organ ratios. The tumour uptake of (111)In-ABY-002 was eightfold higher than that of (111)In-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.

conclusionFor tumours with high HER2 expression, two-helix 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 tumour-to-organ ratios are appreciably higher for three-helix than for two-helix HER2-targeting Affibody molecules.