Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

IntroductionAffibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL).MethodsThe HER2-targeting NCL-cyclized Affibody molecule Z_HER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. The binding affinity of DOTA-Z_HER2:342min was evaluated in vitro. The targeting properties of ¹¹¹In- and ⁶⁸ Ga-DOTA-Z_HER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of ¹¹¹In- and ⁶⁸ Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge.ResultsThe dissociation constant (K_D) for DOTA-Z_HER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-Z_HER2:342min was labeled with ¹¹¹In and ⁶⁸ Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with K_D1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 ± 1.3 for ¹¹¹In- DOTA-Z_HER2:342min and 4.6 ± 0.7 for ⁶⁸ Ga-DOTA-Z_HER2:342min. However, the uptake of DOTA-Z_HER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.ConclusionsNative chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (Z_HER2:342min) with low nanomolar target affinity and specific tumor uptake.