A tool for nuclear imaging of the SARS-CoV-2 entry receptor: molecular model and preclinical development of ACE2-selective radiopeptides

Darja Beyer, Christian Vaccarin, Xavier Deupi, Ana Katrina Mapanao, Susan Cohrs, Fan Sozzi-Guo, Pascal V. Grundler, Nicholas P. van der Meulen, Jinling Wang, Matthias Tanriver, Jeffrey W. Bode, Roger Schibli, Cristina Müller

The angiotensin converting enzyme-2 (ACE2)-entry receptor of SARS-CoV-2-and its homologue, the angiotensin-converting enzyme (ACE), play a pivotal role in maintaining cardiovascular homeostasis. Potential changes in ACE2 expression levels and dynamics after SARS-CoV-2 infection have been barely investigated. The aim of this study was to develop an ACE2-targeting imaging agent as a noninvasive imaging tool to determine ACE2 regulation. DOTA-DX600, NODAGA-DX600 and HBED-CC-DX600 were obtained through custom synthesis and labeled with gallium-67 (T1/2 = 3.26 d) as a surrogate radioisotope for gallium-68 (T1/2 = 68 min). ACE2- and ACE-transfected HEK cells were used for the in vitro evaluation of these radiopeptides. The in vivo tissue distribution profiles of the radiopeptides were assessed in HEK-ACE2 and HEK-ACE xenografted mice and imaging studies were performed using SPECT/CT. The highest molar activity was obtained for [67Ga]Ga-HBED-CC-DX600 (60 MBq/nmol), whereas the labeling efficiency of the other peptides was considerably lower (20 MBq/nmol). The radiopeptides were stable over 24 h in saline (> 99% intact peptide). All radiopeptides showed uptake in HEK-ACE2 cells (36-43%) with moderate ACE2-binding affinity (KD value: 83-113 nM), but no uptake in HEK-ACE cells (< 0.1%) was observed. Accumulation of the radiopeptides was observed in HEK-ACE2 xenografts (11-16% IA/g) at 3 h after injection, but only background signals were seen in HEK-ACE xenografts (< 0.5% IA/g). Renal retention was still high 3 h after injection of [67Ga]Ga-DOTA-DX600 and [67Ga]Ga-NODAGA-DX600 (~ 24% IA/g), but much lower for [67Ga]Ga-HBED-CC-DX600 (7.2 ± 2.2% IA/g). SPECT/CT imaging studies confirmed the most favorable target-to-nontarget ratio for [67Ga]Ga-HBED-CC-DX600. This study demonstrated ACE2 selectivity for all radiopeptides. [67Ga]Ga-HBED-CC-DX600 was revealed as the most promising candidate due to its favorable tissue distribution profile. Importantly, the HBED-CC chelator enabled 67Ga-labeling at high molar activity, which would be essential to obtain images with high signal-to-background contrast to detect (patho)physiological ACE2 expression levels in patients.