The HER2-targeting NCL-cyclized Affibody molecule ZHER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-ZHER2:342min was labeled with 111In and 68 Ga. The binding affinity of DOTA-ZHER2:342min was evaluated in vitro. The targeting properties of 111In- and 68 Ga-DOTA-ZHER2:342min 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.
The dissociation constant (KD) for DOTA-ZHER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-ZHER2:342min was labeled with 111In and 68 Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with KD1 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 ¡À 1.3 for 111In- DOTA-ZHER2:342min and 4.6 ¡À 0.7 for 68 Ga-DOTA-ZHER2:342min. However, the uptake of DOTA-ZHER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts.
Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (ZHER2:342min) with low nanomolar target affinity and specific tumor uptake.