ACE2 and Avidity
The apparent affinity of ACE2 for S incorporates both affinity (site specific binding strength) and avidity (the total sum of all binding interactions). ACE2 is a natural dimer, and dimeric sACE22 is orders of magnitude more effective at neutralizing virus than the monomeric sACE2 protease domain43. The three subunits in a trimeric S spike are conformationally heterogenous, and the RBDs may be exposed for ACE2 interactions in an ‘up’/‘open’ conformation or hidden in a ‘down’/‘closed’ conformation18,77. The dominant conformations of prefusion S trimers are all closed or one RBD in the up state, yet there is a smaller population with additional RBDs accessible for ACE2 interactions78,79,80. There is limited evidence that dimeric ACE22 can bridge two S subunits in a single trimeric spike (i.e. intra-spike avidity)81and atomic structures suggest the spatial orientation of natural dimeric ACE22 is incompatible with binding two RBDs in a single S trimer. Avidity effects are therefore most likely due to dimeric ACE22 cross-linking two separate viral spikes, which is supported by cryoelectron tomography showing moderate spike density on the viral envelope suitable for bridging interactions78,79,80. However, protein engineers are not limited to the geometric architecture of natural ACE2 dimers and have at their disposal tools for creating higher multimeric forms that may sterically complement exposed RBDs on native S trimers (Figure 4).
The creation of sACE2-Fc fusions automatically forms dimeric ACE2 constructs that are superior to monomeric sACE250,81, although we do not foresee any obvious avidity improvements of Fc-mediated dimers over natural dimeric sACE22. By adjusting the fusion site between sACE2 and IgG1 from the hinge to the CH1 and LH domains instead, thereby replacing the variable domains with sACE2, an assembly with four ACE2 chains is created52. This can be further expanded by fusing additional ACE2 moieties to the C-terminus of the H chains to create a hexavalent sACE2-IgG construct, although this arrangement compromised yield52. As expected, avid binding to spikes increases as the oligomeric assemblies get larger.
Trimeric sACE2 that spatially aligns an ACE2 moiety for interactions with each subunit of S in which all RBDs adopt an up conformation will have very high avidity, and has been explored by two groups82,83. The ACE2 protease domain has been fused to trimerization motifs: the C-terminal domain of T4 fibritin (or foldon) and a three helix bundle. The sACE2 trimers can simultaneously bind multiple RBDs in a spike with low picomolar apparent KD. The sACE2-foldon trimer was found to have slightly better inhibitory activity against pseudotyped virus. Affinity-enhancing mutations have also been combined with trimeric sACE2-foldon83.
Lastly, avidity has been increased by fusing an anti-S antibody to sACE2 to create a hybrid, biparatopic ACE2-IgG fusion84. While this protein still contains two ACE2 moieties like other sACE2-IgG fusions, it is distinct in that avidity is also increased by combining bivalent ACE2 binding with a neutralizing antibody that recognizes a non-competing epitope on the spike N-terminal domain. The antibody was selected by yeast display from SARS-CoV-2 survivors. The biparatopic ACE2-IgG hybrid has greatly improved neutralization potency against pseudotyped and authentic virus. Whether this fusion construct compromises serum stability remains to be determined.