ABSTRACT
Background: Soluble Angiotensin Converting Enzyme 2 (ACE2) constitutes an attractive therapeutic candidate with natural resistance to viral escape. To date, ACE2-Fcs, dimeric forms of soluble ACE2, were mostly tested as robust SARS-CoV-2 neutralizers but their potential as antiviral agents capable of Fc-effector functions is largely unknown and has not been tested for effectiveness in vivo, in any model of SARS-CoV2 infection. Methods: We used structure-guided design to select ACE2 mutations that improve SARS-CoV-2 spike (S) affinity and remove angiotensin enzymatic activity. ACE2-Fc variants were engineered into a human IgG1 or IgG3 backbone and produced in mammalian HEK293 cells. S binding was tested by ELISA and surface plasmon resonance (SPR). Mutational effects were validated by X-Ray crystallography. Neutralization activities were measured against SARS-CoV-2 variants of concern (VOCs) using an in vitro pseudovirus (PsV) assay and dynamic bioluminescence imaging (BLI). Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were also quantified using established methods (1, 2). A K18-hACE2 transgenic mouse model challenged by lethal SARS-CoV-2 nLuc infection (3) was used for in vivo evaluation of prophylactic and therapeutic administration of engineered ACE2-Fcs, as monitored by dynamic BLI. Results: Our lead variant, ACE2740 LFMYQY2HA-Fc GASDALIE, increased RBD binding by ∼7-13 fold as compared to wild type, cross-neutralized SARS-CoV-2 VOCs with an IC50 range of 0.23-2.06 nM and mediated robust ADCC and ADCP in vitro. When tested in humanized K18-hACE2 mice, in either a prophylatic or a multi-dosage therapeutic setting, our lead ACE2-Fc variant provided protection from lethal SARS-CoV-2 infection. Our studies in K18-hACE2 mouse model revealed that efficient in vivo efficacy of ACE2-Fcs under prophylaxis or therapeutic settings required Fc-effector functions in addition to neutralization. Conclusion: Our data confirm the utility of engineered ACE2-Fcs as valuable SARS-CoV-2 antivirals and demonstrate that the efficient ACE2-Fc therapeutic activity required both neutralization and Fc-effector functions.