Three Methods for 18F Labeling of the HER2-Binding Affibody Molecule ZHER2:2891 Including Preclinical Assessment

Human epidermal growth factor receptor (HER2)-targeted Affibody molecules radiolabeled with F-18 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 F-18 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. Methods: 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 (F-18-SiFA), F-18-AlF-NOTA, and 4-F-18-fluorobenzaldehyde (F-18-FBA). The biodistributions of the radiolabeled Affibody molecules were then determined in na ve CD-1 nude mice, and tumor targeting was assessed in CD-1 nude mice bearing high-HER2-expressing NCIN87 tumors and low-HER2-expressing A431 tumors. The 111InABY- 025 compound, which has demonstrable clinical utility, served as a reference tracer. Results: The non-decay-corrected radiochemical yields based on starting F-18-fluoride using the F-18-FBA, (18)FSiFA, and F-18-AlF-NOTA methods were 13% 6 3% (n = 5), 38% 6 2% (n = 3), and 11% 6 4% (n 5 6), respectively. In na ve mice, both the F-18-AlF-NOTA-Z(HER2:2891)and the 111In-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 F-18-FBA-ZHER2: 2891 or F-18-SiFAZ(HER2:2891) (4.8 +/- 0.6 and 10.1 +/- 0.7 % ID, respectively, at 90 min). The F-18-SiFA-Z(HER2:2891) conjugate was compromised by increasing bone retention over time (5.3 6 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)FFBA-Z(HER2:2891) in a dual NCI-N87/A431 xenograft model demonstrated high tumor-to-background contrast for NCI-N87 tumors. Conclusion: The HER2 Affibody molecule Z(HER2:2891) has been site-selectively radiolabeled by three F-18 conjugation methods. Preliminary biologic data have identified F-18-FBA-Z(HER2:2891) (also known as GE226) as a favored candidate for further development and radiochemistry automation.