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.