ACE2-based decoy receptors for SARS coronavirus 2

Abstract

SARS coronavirus 2 is neutralized by proteins that block receptor-binding sites on spikes that project from the viral envelope. In particular, substantial research investment has advanced monoclonal antibody therapies to the clinic where they have shown partial efficacy in reducing viral burden and hospitalization. An alternative is to use the host entry receptor, angiotensin-converting enzyme 2 (ACE2), as a soluble decoy that broadly blocks SARS-associated coronaviruses with limited potential for viral escape. Here, we summarize efforts to engineer higher affinity variants of soluble ACE2 that rival the potency of affinity-matured antibodies. Strategies have also been used to increase the valency of ACE2 decoys for avid spike interactions and to improve pharmacokinetics via IgG fusions. Finally, the intrinsic catalytic activity of ACE2 for the turnover of the vasoconstrictor angiotensin II may directly address COVID-19 symptoms and protect against lung and cardiovascular injury, conferring dual mechanisms of action unachievable by monoclonal antibodies. Soluble ACE2 derivatives therefore have the potential to be next generation therapeutics for addressing the immediate needs of the current pandemic and possible future outbreaks.

Keywords: ACE2; COVID-19; SARS coronavirus 2; avidity; decoy receptor; protein engineering.

FIGURE 1

Soluble ACE2 neutralizes SARS‐CoV‐2 infection. Soluble ACE2 (violet) competes with native, membrane‐anchored ACE2 receptors (blue) for binding sites on viral spikes (green). ACE2, angiotensin‐converting enzyme 2; SARS‐CoV‐2, SARS coronavirus 2

FIGURE 2

Selection strategies for enhancing the affinity of ACE2 for the RBD of SARS‐CoV‐2. A, In human cell selections of ACE2 variants, surface expression of full‐length ACE2 with N‐terminal epitope tags was detected via fluorescent anti‐tag antibodies. Cells expressing ACE2 clones with high binding to RBD‐sfGFP were collected by FACS. B, In FACS selections of yeast, the protease domain of ACE2 was expressed as a fusion to Aga2p for display on the yeast surface. Displayed protein was detected via a C‐terminal fusion to GFP or immunostaining of an epitope tag in the connecting linker. Bound RBD was labeled via biotin or an epitope tag for fluorescence detection. C, Structure (PDB 6M17) of RBD (green) bound to ACE2 (pale blue). Common mutations in high‐affinity engineered decoys are indicated in parentheses. D, Mutational landscape showing how single amino acid substitutions in ACE2 increase (blue) or decrease (red) binding to RBD. Data are from Chan et al. ACE2, angiotensin‐converting enzyme 2; FACS, fluorescence activated cell sorting; RBD, receptor‐binding domain; SARS‐CoV‐2, SARS coronavirus 2; sfGFP, superfolder GFP

FIGURE 3

Common modifications to soluble ACE2: fusions with IgG‐Fc and elimination of catalytic activity. A, The Fc region of IgG can be fused to the extracellular domains of ACE2. The Fc moiety is recognized by inflammatory FcγRs on various cell types for immune effector functions. IgG‐Fc also binds FcRn within endosomal compartments following pinocytosis by endothelial and epithelial cells to mediate recycling or transcytosis, impacting biodistribution and serum stability. B, The conversion of angiotensin peptide hormones by renin, ACE1 and ACE2. ACE2, angiotensin‐converting enzyme 2

FIGURE 4

The predicted basis for avid binding of natural ACE2₂ dimers is inter‐spike bridging. Three copies of ACE2₂ dimerized via the collectrin‐like domain (subunits in dark red and blue; PDB 6M17) are overlaid with the structure of trimeric SARS‐CoV‐2 S (subunits are pale shades; PDB 7KMS) with all three RBDs in the up conformation and bound to ACE2 protease domains. Note that in each ACE2₂ dimer, subunit 1 (blue) is bound to a RBD while subunit 2 (red) is directed away from the spike axis where it is accessible for a bridging interaction to another spike (an example is shown as a gray surface at right with a single RBD in the up conformation; PDB 7KNB). Artificial oligomers of sACE2 missing the natural collectrin‐like dimerization domain are not limited to this geometry and might bind with intra‐spike avidity. ACE2, angiotensin‐converting enzyme 2; RBD, receptor‐binding domain; SARS‐CoV‐2, SARS coronavirus 2

FIGURE 5

Strategies to increase sACE2•S avidity. S of SARS‐CoV‐2 binds the monomeric ACE2 protease domain (a.a. 19‐615). A longer version of sACE2 (a.a. 19‐732/740) forms a stable dimer that avidly binds virus. Higher levels of multimerization can be accomplished by various N‐ or C‐terminal fusions to IgG heavy and light chains. Trimeric sACE2 proteins have also been constructed to complement trimeric S spikes. Finally, ACE2 has been fused to an anti‐SARS‐CoV‐2 monoclonal to create an avid biparatopic antibody hybrid. ACE2, angiotensin‐converting enzyme 2; SARS‐CoV‐2, SARS coronavirus 2