Preclinical evaluation of anti-HER2 Affibody molecules site-specifically labeled with ¹¹¹In using a maleimido derivative of NODAGA

Introduction: Affibody molecules have demonstrated potential for radionuclide molecular imaging. The aim of this study was to synthesize and evaluate a maleimido derivative of the 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODAGA) for site-specific labeling of anti-HER2 Affibody molecule. Methods: The maleimidoethylmonoamide NODAGA (MMA-NODAGA) was synthesized and conjugated to Z_HER2:2395 Affibody molecule having a C-terminal cysteine. Labeling efficiency, binding specificity to and cell internalization by HER2-expressing cells of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 were studied. Biodistribution of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 and [¹¹¹In-MMA-DOTA-Cys⁶¹]-Z_HER2:2395 was compared in mice. Results: The affinity of [MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 binding to HER2 was 67 pM. The ¹¹¹In-labeling yield was 99.6%±0.5% after 30 min at 60°C. [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 bound specifically to HER2-expressing cells in vitro and in vivo. Tumor uptake of [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 in mice bearing DU-145 xenografts (4.7%±0.8% ID/g) was lower than uptake of [¹¹¹In-MMA-DOTA-Cys⁶¹]-Z_HER2:2395 (7.5%±1.6% ID/g). However, tumor-to-organ ratios were higher for [¹¹¹In-MMA-NODAGA-Cys⁶¹]-Z_HER2:2395 due to higher clearance rate from normal tissues. Conclusions: MMA-NODAGA is a promising chelator for site-specific labeling of targeting proteins containing unpaired cysteine. Appreciable influence of chelators on targeting properties of Affibody molecules was demonstrated.