Broadly Effective ACE2 Decoy Proteins Protect Mice from Lethal SARS-CoV-2 Infection

ABSTRACT As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been causing increasingly serious drug resistance problem, development of broadly effective and hard-to-escape anti-SARS-CoV-2 agents is an urgent need. Here, we describe further development and characterization of two SARS-CoV-2 receptor decoy proteins, ACE2-Ig-95 and ACE2-Ig-105/106. We found that both proteins had potent and robust in vitro neutralization activities against diverse SARS-CoV-2 variants, including BQ.1 and XBB.1, that are resistant to most clinically used monoclonal antibodies. In a stringent lethal SARS-CoV-2 infection mouse model, both proteins lowered the lung viral load by up to ~1,000-fold, prevented the emergence of clinical signs in >75% animals, and increased the animal survival rate from 0% (untreated) to >87.5% (treated). These results demonstrate that both proteins are good drug candidates for protecting animals from severe COVID-19. In a head-to-head comparison of these two proteins with five previously described ACE2-Ig constructs, we found that two constructs, each carrying five surface mutations in the ACE2 region, had partial loss of neutralization potency against three SARS-CoV-2 variants. These data suggest that extensively mutating ACE2 residues near the receptor binding domain (RBD)-binding interface should be avoided or performed with extra caution. Furthermore, we found that both ACE2-Ig-95 and ACE2-Ig-105/106 could be produced to the level of grams per liter, demonstrating the developability of them as biologic drug candidates. Stress condition stability testing of them further suggests that more studies are required in the future to improve the stability of these proteins. These studies provide useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. IMPORTANCE Engineering soluble ACE2 proteins that function as a receptor decoy to block SARS-CoV-2 infection is a very attractive approach to creating broadly effective and hard-to-escape anti-SARS-CoV-2 agents. This article describes development of two antibody-like soluble ACE2 proteins that broadly block diverse SARS-CoV-2 variants, including Omicron. In a stringent COVID-19 mouse model, both proteins successfully protected >87.5% animals from lethal SARS-CoV-2 infection. In addition, a head-to-head comparison of the two constructs developed in this study with five previously described ACE2 decoy constructs was performed here. Two previously described constructs with relatively more ACE2 surface mutations were found with less robust neutralization activities against diverse SARS-CoV-2 variants. Furthermore, the developability of the two proteins as biologic drug candidates was also assessed here. This study provides two broad anti-SARS-CoV-2 drug candidates and useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses.