Optimized Molecular Design of ADAPT-Based HER2-Imaging Probes Labeled with ¹¹¹In and ⁶⁸Ga

Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of ¹¹¹In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)₃DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C⁵⁹-DEAVDANS-ADAPT6-GSSC and DOTA-C⁶¹-(HE)₃DANS-ADAPT6-GSSC) were stably labeled with ¹¹¹In for SPECT and ⁶⁸Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. ¹¹¹In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to ⁶⁸Ga-labeled counterparts. The best performing variant was DOTA-C⁶¹-(HE)₃DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for ¹¹¹In and ⁶⁸Ga labels, respectively.