The spike protein (S) of SARS coronavirus 2, the pathogen responsible for COVID-19, binds angiotensin-converting enzyme 2 (ACE2) as an entry receptor, triggering conformational changes in S that drive fusion ofthe viral envelope and host cell membrane. Infection is inhibited by neutralizing antibodies that block theACE2-binding site on S, yet escape mutations in S rapidly emerge towards monoclonal antibodies in tissueculture. Furthermore, monoclonal antibodies are generally strain specific, and many do not recognize with highaffinity both human SARS-CoV-1 and SARS-CoV-2, yet alone exotic bat betacoronaviruses that are a reservoirfor future outbreaks. As an alternative for biologic drug development, we have used deep mutagenesis toguide the engineering of an exceptionally broad soluble decoy receptor that binds with tight picomolar/low-nanomolar affinity to S from all bat and human SARS-associated coronaviruses tested. The engineered decoypotently neutralizes authentic SARS-CoV-1 and SARS-CoV-2 with an efficacy that rivals monoclonals undercommercial development, and has desirable properties for manufacture at scale. The engineered decoy alsocatalytically converts angiotensin II to vasodilatory peptide products that might directly address symptoms ofCOVID-19, providing us with a unique potential therapeutic that has dual mechanisms of action. Our proposalinvestigates whether the SARS-CoV-2 spike can mutate to escape neutralization by the engineered decoyreceptor, and addresses final optimization of the engineered protein as an IgG1-Fc fusion before advancementto an IND-enabling program. For SARS-CoV-2 to become resistant to the engineered decoy, mutations in Smust decrease affinity to the decoy while maintaining binding to human ACE2 receptors. To identify such Svariants, we have used saturation mutagenesis of the receptor-binding domain coupled with a selection fortight binding to wild type ACE2 in the presence of competing soluble decoy. Following deep sequencing, asmall number of mutations were found to be enriched, but it is unclear whether any of these mutations doindeed preferentially bind wild type ACE2 and if so, to what degree they have achieved specificity. Based onthis preliminary data, (Aim 1) we will validate whether mutations in S can be found that discriminate betweenhuman ACE2 and the engineered decoy, and characterize the variants for their affinities and expression levels.Thus far, we can conclude that possible resistance mutations appear to be very rare and generally requiremore than one nucleotide change within a codon, but further quantitative characterization is needed.Simultaneously, (Aim 2) we will rapidly optimize fusions of the engineered decoy with the Fc region of IgG1 forenhanced serum stability. Our current IgG1-fusion construct (which was based on rational, structure-guideddesign) is highly expressed, stable and binds SARS-CoV-2 S with picomolar affinity. We will finalizeoptimization of the protein by scanning suitable fusion sites between the engineered decoy and IgG1,assessing protein quality by activity, stability and expression. NARRATIVE
The receptor for SARS-CoV-2 has been engineered as a high-affinity soluble decoy that potently neutralizes
authentic virus and has broad activity against SARS-associated coronaviruses that use ACE2 as an entry
receptor. We propose to use deep mutagenesis to identify possible spike variants that may preferentially bind
wild type ACE2 rather than the engineered decoy, and as such may be a source for escape mutations and
resistance. We further propose to optimize a fusion between the engineered decoy and the Fc region of IgG1
for a serum stable biologic suitable for advancing towards an IND application. 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