Three methods for 18F labeling of the HER2-binding affibody molecule Z(HER2:2891) including preclinical assessment

Human epidermal growth factor receptor (HER2)-targeted Affibody molecules radiolabeled with (18)F allow the noninvasive assessment of HER2 status in vivo through PET imaging. Such agents have the potential to improve patient management by selecting individuals for HER2-targeted therapies and allowing therapy monitoring. The aim of this study was to assess different (18)F radiolabeling strategies of the HER2-specific Affibody molecule Z(HER2:2891), preclinically determine the biologic efficacy of the different radiolabel molecules, and select a preferred radiolabeling strategy to progress for automated manufacture.

Cysteine was added to the C terminus of the Affibody molecule for the coupling of maleimide linkers, and 3 radiolabeling strategies were assessed: silicon-fluoride acceptor approach ((18)F-SiFA), (18)F-AlF-NOTA, and 4-(18)F-fluorobenzaldehyde ((18)F-FBA). The biodistributions of the radiolabeled Affibody molecules were then determined in naïve CD-1 nude mice, and tumor targeting was assessed in CD-1 nude mice bearing high-HER2-expressing NCI-N87 tumors and low-HER2-expressing A431 tumors. The (111)In-ABY-025 compound, which has demonstrable clinical utility, served as a reference tracer.

The non-decay-corrected radiochemical yields based on starting (18)F-fluoride using the (18)F-FBA, (18)F-SiFA, and (18)F-AlF-NOTA methods were 13% ± 3% (n = 5), 38% ± 2% (n = 3), and 11% ± 4% (n = 6), respectively. In naïve mice, both the (18)F-AlF-NOTA-Z(HER2:2891) and the (111)In-ABY-025 compounds showed a significant kidney retention (70.3 ± 1.3 and 73.8 ± 3.0 percentage injected dose [%ID], respectively, at 90 min after injection), which was not observed for (18)F-FBA-Z(HER2:2891) or (18)F-SiFA-Z(HER2:2891) (4.8 ± 0.6 and 10.1 ± 0.7 %ID, respectively, at 90 min). The (18)F-SiFA-Z(HER2:2891) conjugate was compromised by increasing bone retention over time (5.3 ± 1.0 %ID/g at 90 min after injection), indicating defluorination. All the radiolabeled Affibody molecules assessed showed significantly higher retention in NCI-N87 tumors than A431 tumors at all time points (P < 0.05), and PET/CT imaging of (18)F-FBA-Z(HER2:2891) in a dual NCI-N87/A431 xenograft model demonstrated high tumor-to-background contrast for NCI-N87 tumors.

The HER2 Affibody molecule Z(HER2:2891) has been site-selectively radiolabeled by three (18)F conjugation methods. Preliminary biologic data have identified (18)F-FBA-Z(HER2:2891) (also known as GE226) as a favored candidate for further development and radiochemistry automation.