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.
ABSTRACT
Background: Both neutralizing activity and Fc-mediated effector functions of antibodies are believed to contribute to protection against SARS-CoV-2. However, it is unclear if antibody effector functions alone could protect against SARS-CoV-2 infection. Methods: We isolated CV3-13 from a convalescent individual with potent Fc-mediated effector functions. Neutralization capacity of this antibody was measured by both a pseudovirus neutralization assay and an authentic virus microneutralization assay. We mutated the Fc-portion of CV3-13 to enhance (GASDALIE) or reduce (LALA) its capacity to mediate antibody dependant cellular cytotoxicity (ADCC). Structural analysis of CV3-13 was done by cryo-EM to characterize its epitope and its angle of approach. Finally, CV3-13 and CV3-13 GASDALIE were used in vivo in a K18-hACE2 transgenic mouse model challenged with SARS-CoV-2-nLuc to see if they altered viral replication and/or contributed to protection against SARS-CoV-2. Results: While CV3-13 did not neutralize SARS-CoV-2, it demonstrated nanomolar affinity towards the SARS-CoV-2 Spike and mediated strong ADCC. The cryo-EM structure of CV3-13 in complex with the SARS-CoV-2 Spike revealed that the antibody bound to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. Interestingly, this angle of approach was not observed for previously described NTD-directed antibodies. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, a Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Conclusion: CV3-13 represents a new class of non-neutralizing NTD-directed mAbs that can mediate Fc-effector functions both in vitro and in vivo. While effector functions alone did not protect K18-hACE2 mice from SARS-CoV-2-nLuc challenge, our data indicate that along with neutralization, additional antibody properties including Fc-mediated effector functions contribute to limiting viral spread and aid in fighting SARS-CoV-2 infection.