\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[T2A]{fontenc}
\usepackage[ngerman,greek,polish,english]{babel}



\usepackage{float}








\begin{document}

\title{ACE2-Based Decoy Receptors for SARS Coronavirus 2}



\author[1]{Wenyang Jing}%
\author[1]{Erik Procko}%
\affil[1]{University of Illinois at Urbana-Champaign}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
SARS-CoV-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 there are signs of partial efficacy in
reducing viral burden and hospitalization. An alternative is to use the
host entry receptor, 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.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Not long following its emergence in China in late 2019, SARS coronavirus
2 (SARS-CoV-2) became an unprecedented public health emergency for our
generation. The absence of immunity, superspreader
events\textsuperscript{1} and presymptomatic\textsuperscript{2} and
asymptomatic\textsuperscript{3,4} transmission have all combined to
favor this respiratory virus' global spread. The virus is devastating to
the elderly and other vulnerable groups with certain predisposing
conditions, and elicits surprisingly heterogenous disease symptoms
collectively known as COVID-19, with the most common being fever, dry
cough, hypoxemia and pneumonia\textsuperscript{5,6,7,8}, but also
unusual neurological symptoms\textsuperscript{9} and
coagulopathy\textsuperscript{10}. It remains unclear which aspects of
disease are the result of disseminated virus infection of multiple
tissues versus dysregulation of signaling pathways, including cytokine
storms\textsuperscript{11,12} and aberrant angiotensin and kinin peptide
processing\textsuperscript{13,14,15}.

Virus entry is mediated by interactions between the viral spike, a
trimeric complex of protein S, and angiotensin-converting enzyme 2
(ACE2) on a host cell membrane\textsuperscript{16,17,18,19,20,21,22,23}.
S is proteolytically processed as two subunits, S1 and S2, that remain
noncovalently associated until ACE2 is bound by a receptor-binding
domain (RBD) in the S1 subunit, triggering conformational changes that
include S1 shedding, exposure of a fusion peptide at the N-terminus of
S2 and fusion of the viral envelope with the host
membrane\textsuperscript{21,24,25,26,27}. Antibodies targeting multiple
epitopes on S, but especially the RBD, can block ACE2 engagement or
prevent membrane fusion from occurring, and numerous monoclonal antibody
therapies are now in preclinical and clinical
development\textsuperscript{28,29,30,31,32,33}. However, coronaviruses
have moderate to high mutation rates\textsuperscript{34,35} and there is
a perceived risk that drug resistant SARS-CoV-2 variants might begin
circulating once antibody therapies become widely used. This is
addressed by combining noncompeting monoclonal antibodies as
cocktails\textsuperscript{36}.

An alternative strategy is to use ACE2 itself as a soluble decoy
receptor that competes for receptor-binding sites on
S\textsuperscript{22,37,38,39} (Figure 1). ACE2 is an 805 amino acid
(a.a.) protein that comprises a protease domain (a.a. 19-615), a
collectrin-like dimerization domain (a.a. 616-729) and a single-span
transmembrane domain (a.a. 741-765)\textsuperscript{16}. The major
attraction of using an entry receptor as a soluble decoy is that, in
principle, the virus has limited mutational mechanisms for escape
without simultaneously losing affinity for the native, membrane anchored
form\textsuperscript{40}. Soluble decoy receptors are used clinically
for a variety of indications, although none are yet approved drugs for
viruses\textsuperscript{41}. Wild type, soluble ACE2 (sACE2) is an
investigational drug for acute respiratory distress that has been
rapidly repurposed as a SARS-CoV-2 antiviral\textsuperscript{37} and has
entered a phase II COVID-19 clinical trial managed by Apeiron
Biologics\textsuperscript{42}. This drug candidate has become the
starting point for multiple engineering efforts to solve key issues
surrounding pharmacokinetics, affinity and avidity for the creation of
next generation ACE2 derivatives with superior efficacy. It is these
efforts that are reviewed here.

\textbf{Mutagenesis of ACE2 for Enhanced Affinity}

Soluble decoy receptors should ideally bind the SARS-CoV-2 spike tighter
than, and out-compete, the wild type (WT) ACE2 receptor. Tight affinity
is also necessary for effective virus neutralization at typical doses
for biologic drugs. The importance of affinity is further emphasized
when soluble decoys are considered against the therapeutic alternative
of affinity-matured monoclonal antibodies, which bind S of SARS-CoV-2
with tight nanomolar to picomolar
K\textsubscript{D}\textsuperscript{28,29,30,33}; by comparison, WT sACE2
has only moderate affinity (KD [?] 20 nM)\textsuperscript{16,43}. To this
end, mutagenic approaches, either targeted or through selection of
diverse libraries of variants, provide a means for engineering decoy
receptors with enhanced affinity.

In the first report\textsuperscript{44} that mutations within ACE2 can
indeed be found that increase S affinity, Chan et al. performed deep
mutagenesis on full length ACE2\textsuperscript{43,45}. A library of
ACE2 variants was created that encoded all possible single amino acid
variants at 117 sites, covering both the regions that interface with the
RBD as well as the ACE2 active site. The library was then selected by
fluorescence activated cell sorting (FACS) for ACE2 surface expression
in human cells and tight binding to the RBD of SARS-CoV-2 protein S
(Figure 2). ACE2 residues buried at the interface with S tend to be more
conserved, while ACE2 residues at the interface periphery or within the
active site were mutationally tolerant. Mutations of ACE2 residues N90
and T92, which form an N-glycosylation motif, are universally enriched
for high binding affinity (with the exception of T92S), indicating that
N90 glycosylation hinders binding, at least in this experiment's cell
line. Other mutations were also found dispersed across multiple sites
that increase S binding, and some of these were rapidly screened as
combinations of mutations in soluble ACE2 conjugated to superfolder GFP
(sfGFP)\textsuperscript{46}. Expression medium containing sACE2-sfGFP,
without purification, can be incubated directly with cells expressing S
and bound sACE2-sfGFP detected by flow cytometry. Ultimately, variants
of sACE2 carrying four or three mutations (Figure 2C), dubbed sACE2.v2
and sACE2.v2.4, were engineered for an optimal balance of high affinity,
expression and yield. Impressively, monomeric sACE2.v2, which spans ACE2
residues 19-615 that form the extracellular protease domain, outcompeted
WT sACE2 fused to dimeric Fc of IgG1 and also competed effectively with
anti-RBD Ig derived from Covid-19 positive patient sera. Soluble ACE2
forms a natural dimer (which we refer to as sACE2\textsubscript{2}) if
the construct is expanded up to ACE2 residues 730-740, which includes
the dimerization/collectrin-like domain immediately C-terminal of the
protease domain. Soluble ACE2\textsubscript{2} has increased apparent
affinity and neutralization of authentic virus through avidity,
demonstrating that each protease domain in sACE2\textsubscript{2} can
bridge two S proteins. The leading variant from these efforts, dimeric
sACE2\textsubscript{2}.v2.4, had a picomolar K\textsubscript{D} for the
SARS-CoV-2 RBD and achieved 50\% relative infection inhibition of
authentic virus at subnanomolar concentrations. Surprisingly, it showed
similar neutralization efficacy against SARS-CoV-1 despite no
consideration of binding to this virus during mutagenesis. This implied
potential efficacy against other zoonotic coronaviruses that utilize
ACE2 and may spill over to humans in the future. This was confirmed by
demonstrating sACE2\textsubscript{2}.v2.4 tightly binds diverse RBD
sequences from SARS-related bat betacoronaviruses that use ACE2 as an
entry receptor\textsuperscript{40}. Furthermore, an in vitro selection
of mutations within the RBD from SARS-CoV-2 failed to find S variants
that lose affinity for the engineered sACE2\textsubscript{2}.v2.4 decoy
but retain binding to the wild type host ACE2 receptor, thus confirming
the hypothesis that mechanisms for viruses to escape soluble decoy
receptors are limited\textsuperscript{40}.

In a similar deep mutagenesis experiment, Heinzelman and Romero
investigated the influence on S protein binding of single-nucleotide
variants (SNVs) in the ACE2 protease domain, evaluating nearly 4,000
amino acid substitutions\textsuperscript{47}. Mutations were made using
error-prone PCR, a rapid and reliable way of generating library
diversity but which fails to capture multiple nucleotide changes within
a single codon (unless error rates are excessively high). The library of
the ACE2 protease domain was screened by yeast surface display (Figure
2B), and while most mutations decreased binding (it is, after all, easy
to 'break' a protein through random mutagenesis), approximately 4\% of
mutations were found to increase RBD binding. While mutations in the
active site tended to have little effect (with the caveat that the
background ACE2 sequence used for library generation already had
catalytic activity knocked out), the authors found that residues in the
chloride-binding site of ACE2, which regulates its peptidase
activity\textsuperscript{48} and is over 40 \selectlanguage{ngerman}Å from the RBD interface, do
affect binding. In addition, a separate distal cluster of hydrophobic
residues (L236, F588, L591, and L595) was also identified as a critical
site affecting binding despite being over 30 Å from the RBD interface.
By interrogating SNVs, the authors were also able to statistically
evaluate the clinical and epidemiological relevance of ACE2 allelic
variants in the human population, and they estimated that roughly 1 in
10,000 people may have SNVs that increase spike binding and roughly 4 in
1000 having SNVs that decrease spike binding. However, these predictions
will need to be confirmed by genetics studies of patient cohorts,
especially since mutations in ACE2 with altered affinity for S may also
have other effects that impact infection in vivo, such as expression
changes in human tissues. Overall, this work highlights the importance
of considering long-range effects of mutations distal from the
functional binding site.

The deep mutational scans of ACE2 are based on a single round of
sequence diversification and selection, whereas others have employed
multiple rounds to direct the in vitro evolution of ACE2 towards
exceptionally tight affinity for SARS-CoV-2 S. Higuchi et al. focused
mutagenesis to ACE2 residues 18-102 and 272-409 in the protease domain,
respectively denoted as PD1 and PD2, that form the RBD
interface\textsuperscript{49}. Binding was assayed using the method
previously developed for deep mutagenesis\textsuperscript{43}, with
human cells expressing the ACE2 library incubated with RBD-sfGFP and
sorted by FACS. Mutations within the ''PD1'' region were selected first,
followed by mutagenesis within the ''PD2'' region and additional rounds
of selection, although additional mutations within the PD2 region did
not substantially improve affinity. The ACE2 variant identified with
highest affinity, termed 3N39 with 7 substitutions (Figure 2C), bound
RBD with picomolar K\textsubscript{D}. Despite having a much higher
mutational load than sACE2.v2.4, soluble 3N39-Fc fusions were soluble
with minimal aggregation by size exclusion chromatography. The
engineered 3N39-Fc fusion vastly outperformed the equivalent wild type
ACE2-Fc fusion in virus neutralization.

The Kortemme, Hobman, and Wells groups went even further in using
multiple rounds of engineering, diversification and selection to drive
down the dissociation constant to the picomolar
range\textsuperscript{50}. Computational alanine scanning identified
ACE2 residues H34, Q42 and K353 as hot spots that contribute
disproportionately to the binding energy, and these residues with their
neighbors were the focus for computational mutagenesis. Computationally
designed ACE2 variants were purified as Fc fusions and binding to S was
improved 3- to 11-fold. Four of these ACE2 variants served as parents
that were then matured through yeast surface display, resulting in a
further 14-fold improvement in apparent K\textsubscript{D}. Deep
mutagenesis data from Chan et al\textsuperscript{43} was then
considered, from which new ACE2 variants were designed with mutations
that were not alanine scan-based hotspots and included sites outside the
RBD interface that likely stabilize conformation. The final ACE2
variants had exceptionally tight affinity, with the lowest
K\textsubscript{D} variants carrying 4-8 substitutions (Figure 2C). It
was also independently confirmed that additional increases in apparent
affinity can be achieved through inclusion of the ACE2 dimerization
domain for avid binding and protein stabilization. It is worth noting
that in this study deliberate action was taken to inactivate ACE2
enzymatic activity. This has historically been done by mutating two
critical Zn\textsuperscript{2+}-coordinating residues (H374N and
H378N)\textsuperscript{51}, but because these substitutions resulted in
undesirable destabilization in the variant background, an alternative
mutation (H345L within the substrate-binding cavity) was adopted. While
the rationale for this is the elimination of vasodilatory effects from
excess ACE2 peptidase activity that may cause hypotension, it is worth
considering the potential benefits of keeping enzymatic activity
(discussed below).

Pangolin (Pangolin-CoV-2020) and bat (isolate RaTG13) coronaviruses, in
addition to human SARS-CoV-1 and -2, can initiate membrane fusion using
not only human ACE2 but many other animal orthologs\textsuperscript{52}.
This informed the substitution of human ACE2-D30 for
glutamate\textsuperscript{52}, which is found in ACE2 of other species
and is able to better reach K417 of the RBD for salt bridge
formation\textsuperscript{16,17}. In addition, inclusion of the
dimerization domain together with the protease domain further
facilitated the engineering of a sACE2-Ig fusion protein effective
against multiple betacoronavirus strains. The discovery of the
affinity-enhancing D30E mutation and benefits of including the
dimerization domain overlap with independent work by
others\textsuperscript{43,50}. Another contributing factor was increased
avidity (to be discussed further below) due to design of a tetrameric
sACE2-Ig configuration\textsuperscript{52}. Overall, it is apparent from
all these studies that a truly effective therapeutic approach utilizing
decoy ACE2 receptors will likely incorporate a multifaceted strategy of
affinity enrichment, avidity and Fc fusions.

One final aspect to touch upon regarding affinity enhancement through
mutagenesis is the connection between binding affinity and ACE2
enzymatic activity. For example, the sACE2.v2.4 variant has reduced
catalytic activity even though its 3 amino acid substitutions are not
within the active site\textsuperscript{43}, thus demonstrating
potentially unappreciated coupling between S affinity and ACE2
catalysis. Others have observed that some mutations that knock out ACE2
activity can increase S binding\textsuperscript{53}, although many
mutations in the active site have no effect on S
affinity\textsuperscript{43,47}. In particular, we highlight again the
key finding by Heinzelman and Romero that mutations to a
chloride-binding motif of ACE2, which is 40 Å from the RBD interface and
is directly involved in regulating peptidase activity, can affect spike
affinity, possibly due to conformational effects\textsuperscript{47}.
The binding of trimeric SARS-CoV-2 spike protein to wild type ACE2 also
increases its peptidase activity 3- to 10-fold, with the SARS-CoV-2 RBD
inducing bending of the N-terminal protease subdomain toward the
C-terminal protease subdomain ``\ldots{}reminiscent of a closing
dam''\textsuperscript{54}. Although the precise mechanisms remain
unclear, there are nonetheless multiple clues incriminating ACE2
conformation and dynamics on both catalytic activity and S affinity. We
believe from a therapeutic perspective (to be discussed further below),
maintaining catalytic activity is preferable due to the protective
effects of ACE2 products on the pulmonary and cardiovascular systems.

\textbf{ACE2 Fusions with Immunoglobulin Fc}

Despite an ongoing clinical trial of wild type sACE2 in
Europe\textsuperscript{42} and the studies reviewed above demonstrating
the realization of picomolar affinity decoy receptors, it is likely that
low sACE2 serum stability will limit its successful application as a
therapeutic. The serum half-life of dimeric sACE2 in vivo is on the
order of hours\textsuperscript{55,56}, thereby requiring frequent dosing
to maintain sACE2 in circulation. As a consequence, it is questionable
whether sACE2 can be safely administered in an outpatient setting. Serum
stability of biologic drugs is greatly increased by fusion of the
therapeutic protein with the Fc region of IgG\textsuperscript{57}. Serum
proteins are turned over by constant internalization and lysosomal
degradation, especially by endothelial cells. Internalized IgG-Fc
engages the neonatal Fc receptor (FcRn) within endosomal compartments to
direct its trafficking back to the plasma membrane, thereby returning
the protein to the serum (Figure 3A). The previously highlighted works
describing the discovery of high affinity sACE2
variants\textsuperscript{43,49,50,52} have already extended their
findings to fusions with IgG1 Fc, and we believe that sACE2-Fc fusions
will be the standard going forward for the production of high-affinity
decoy receptors with excellent pharmacokinetics.

Recombinant human sACE2 has been developed over nearly two decades as a
therapeutic for lung inflammation and acute respiratory distress
syndrome (ARDS). Its administration has been shown to be protective
against cardiovascular\textsuperscript{58} and pulmonary pathologies and
to treat hypoxemia in multiple animal
models\textsuperscript{59,60,61,62,63}, and it has acceptable safety
without causing episodes of hypotension in healthy and mechanically
ventilated human subjects\textsuperscript{56,64}. However, while sACE2
demonstrated the expected in vivo biochemical activity in ARDS patients,
there was no statistically significant improvement in clinical outcomes
in a phase 2 trial. The clinicians commented that ``\ldots{}lower plasma
concentrations (of sACE2) over a longer duration may be more effective
as a result of more sustained production of'' ACE2
products\textsuperscript{64}. These findings motivated the first
published report of an sACE2-Fc fusion tested in vivo (in mice), in
which the serum half-life was extended to over a week with substantially
higher maximum serum activity\textsuperscript{63}. Recombinant sACE2-Fc
mitigated hypertension following injection of the vasopressor (and ACE2
substrate) angiotensin II for up to 7 days, whereas unfused sACE2 lost
function in serum within hours. The Fc fusion protein reduced blood
pressure and cardiac and renal fibrosis in a transgenic mouse model for
hypertension and albuminuria in which renin is massively
overexpressed\textsuperscript{63}. This work demonstrated serum
longevity of sACE2 fused to IgG1-Fc as well as the potent physiological
effects of ACE2 catalytic activity.

More recently with the emergence of SARS-CoV-2, multiple groups have
explored IgG1-Fc fusions of sACE2 and consistently find long serum
half-life without adverse toxicity in small
animals\textsuperscript{53,38}. The Fc region of IgG subclasses engage
multiple Fc receptors to effect function, including not only FcRn to
mediate serum stability and uptake into the lungs, but also a suite of
inflammatory Fc \selectlanguage{greek}γ \selectlanguage{english}receptors (Fc\selectlanguage{greek}γ\selectlanguage{english}R)\textsuperscript{65}. These receptors
are expressed on a variety of cell types, especially in the immune
system, and are responsible for effector functions such as
antibody-dependent cellular cytotoxicity, cytokine release, antigen
presentation and co-stimulation (Figure 3A). Until different Fc fusion
proteins are properly assessed in controlled in vivo experiments, it is
unclear whether these effector functions will aid in clearance and
recovery from SARS-CoV-2 infection. Iwanaga et al have hypothesized that
interactions of a sACE2-Fc biologic with inflammatory Fc\selectlanguage{greek}γ\selectlanguage{english}Rs may
exacerbate inflammation and cytokine storms in COVID-19
patients\textsuperscript{53}. Consequently, two leucine residues in the
IgG1-Fc moiety were mutated to alanines (L234A and L235A). These
so-called LALA mutations disrupt Fc\selectlanguage{greek}γ\selectlanguage{english}R interactions while leaving FcRn
binding intact for high serum stability. The LALA variant of sACE2-Fc
had long serum half-life, was distributed to lung tissues, and retained
virus neutralization efficacy.

\textbf{Catalytically active or inactive sACE2?}

Most reports describing the engineering of sACE2 to enhance its affinity
through targeted substitutions, or to improve pharmacokinetics through
Fc fusions, have deliberately eliminated ACE2 catalytic
activity\textsuperscript{50,53,38}. The assumption is that
administration of a catalytically active decoy receptor will adversely
interact with physiology to have unacceptable toxicity. Soluble decoy
receptors for other viruses have also been engineered to eliminate the
normal biological activity for safety and efficacy
reasons\textsuperscript{66}. However, there are strong arguments that
ACE2 catalytic activity, even if elevated, might be beneficial for
treating COVID-19 symptoms.

In the renin-angiotensin system (RAS), a set of proteases regulates the
production of angiotensin peptide hormones\textsuperscript{67,68}(Figure
3B). Renin is released by the kidneys when renal blood flow is low and
it catalyzes the conversion of angiotensinogen in the blood to
angiotensin I (Ang-I). Ang-I is in turn a substrate for
angiotensin-converting enzyme (ACE or ACE1), which cleaves the peptide
to produce the vasoconstrictor Ang-II that has a wide range of effects
through the angiotensin II type 1 receptor (AT\textsubscript{1}R).
Ang-II promotes vasoconstriction and elevated blood pressure and volume,
and under conditions where Ang-II is high there can be increased
inflammation, myocardial fibrosis and thrombosis. Ang-II is proteolyzed
by ACE2 to produce Ang-1-7\textsuperscript{69,70,71,72}, which has
vasodilatory properties and is cardioprotective\textsuperscript{73}.
Both ACE1 and ACE2 are active in lung tissue (and elsewhere) and also
catalyze the conversion of kinin peptides, the consequences of which are
still speculative in the context of COVID-19\textsuperscript{15,74}.
Genetic studies and administration of sACE2 in animal models have
demonstrated that tilting the RAS system in favor of Ang-1-7 has
numerous benefits for the treatment of lung injury and
inflammation\textsuperscript{59,60,61}. Indeed, it has been proposed
that COVID-19 symptoms may in part manifest from RAS
dysregulation\textsuperscript{37,68}. Ang-II levels are elevated
approximately 3-fold in hospitalized COVID-19 patients and are
correlated with both viral load and severity of hypoxemic respiratory
failure\textsuperscript{13}. Catalytically active sACE2 therefore offers
two potential mechanisms of action. First, it blocks receptor-binding
sites on SARS-CoV-2 spikes to neutralize infection. Second, it promotes
the degradation of Ang-II and production of Ang-1-7 to directly address
COVID-19 symptoms. A clinical trial testing wild type sACE2 in
critically ill COVID-19 patients is ongoing, but a preliminary case
report offers promising signs that both mechanisms of action are at
play\textsuperscript{42}. This provides sACE2 a tremendous advantage
over monoclonal antibodies, which are safe and effective at reducing
viral load\textsuperscript{75} but do not directly address symptoms in
sick patients. However, at this time no direct comparison has been made
in animal models of COVID-19 to evaluate catalytically active versus
inactive sACE2, alongside engineered variants with altered S affinity.
The hypothesis that sACE2 will have dual mechanisms of action therefore
remains unproven.

Different mutations have been introduced into sACE2 to eliminate
catalytic activity, the most common being H374N and H378N to prevent
coordination of an essential
Zn\textsuperscript{2+}ion\textsuperscript{51}. Others have used
alternative mutations within the ACE2 active
site\textsuperscript{50,53}. As a generalization, ACE2 variants with
reduced catalytic activity bind slightly tighter to SARS-CoV-2 S,
suggesting possible conformational coupling between the catalytic and S
binding sites. We are aware of only a single report\textsuperscript{76}
where mutations to inactivate catalysis were associated with a small
decrease in neutralization efficacy.

\textbf{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
sACE2\textsubscript{2} is orders of magnitude more effective at
neutralizing virus than the monomeric sACE2 protease
domain\textsuperscript{43}. 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' conformation\textsuperscript{18,77}. 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 interactions\textsuperscript{78,79,80}. There is limited
evidence that dimeric ACE2\textsubscript{2} can bridge two S subunits in
a single trimeric spike (i.e. intra-spike
avidity)\textsuperscript{81}and atomic structures suggest the spatial
orientation of natural dimeric ACE2\textsubscript{2} is incompatible
with binding two RBDs in a single S trimer. Avidity effects are
therefore most likely due to dimeric ACE2\textsubscript{2} cross-linking
two separate viral spikes, which is supported by cryoelectron tomography
showing moderate spike density on the viral envelope suitable for
bridging interactions\textsuperscript{78,79,80}. 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 sACE2\textsuperscript{50,81},
although we do not foresee any obvious avidity improvements of
Fc-mediated dimers over natural dimeric sACE2\textsubscript{2}. By
adjusting the fusion site between sACE2 and IgG1 from the hinge to the
C\textsubscript{H}1 and L\textsubscript{H} domains instead, thereby
replacing the variable domains with sACE2, an assembly with four ACE2
chains is created\textsuperscript{52}. 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 yield\textsuperscript{52}. 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
groups\textsuperscript{82,83}. 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
K\textsubscript{D}. 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-foldon\textsuperscript{83}.

Lastly, avidity has been increased by fusing an anti-S antibody to sACE2
to create a hybrid, biparatopic ACE2-IgG fusion\textsuperscript{84}.
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.

\textbf{Vesicle-Based ACE2 Nanoparticle Decoys}

While this review has focused on soluble ACE2 receptors and their
associated Ig-fusions, work has also been done utilizing
membrane-anchored ACE2. These decoys are based on cell-derived vesicles
or fabricated nanoparticles derived from cells expressing
membrane-embedded ACE2\textsuperscript{85,86,87}. Extracellular vesicles
produced from cells over-expressing ACE2 together with TMPRSS2---which
is required for membrane fusion following ACE2/S
binding\textsuperscript{21}---were more effective at neutralizing
pseudotyped viruses than purified sACE2, perhaps because of inbuilt
avidity from high density receptor display\textsuperscript{86}. TMPRSS2
was required for optimum activity, and it is possible bound viruses
fused with the vesicles, effectively creating irreversible
neutralization. Vesicles have been further functionalized by the use of
membranes from monocytes that naturally express cytokine receptors, such
that both virus and cytokines are sequestered on the nanoparticle
surface\textsuperscript{87}. Inhibited cytokine signaling might
ameliorate potentially life-threatening inflammatory responses
associated with severe COVID-19. Cytokine neutralization activity was
confirmed in vivo by suppressed inflammation in a
lipopolysaccharide-induced lung injury model in mice. While
vesicle-based nanoparticles are scientifically exciting and show
efficacy for virus and cytokine neutralization, they will almost
certainly suffer a tortuous regulatory pathway to the clinic due to
composition heterogeneity and safety concerns.

\textbf{Conclusion}

Emergency Use Authorization from the U.S. Food and Drug Administration
is currently being sought for two monoclonal antibody therapies for
COVID-19, with others under development. It has been conclusively
demonstrated that soluble ACE2 decoys can be engineered to rival the
affinities of monoclonal antibodies, and provided IgG fusions confer
desirable pharmacokinetics in humans, there is no reason to expect
soluble ACE2 decoys cannot be as effective as monoclonals in reducing
viral load in patients. Anti-SARS-CoV-2 monoclonals have received a
preferential regulatory environment for clinical advancement due to an
in-depth understanding of antibody biochemistry, pharmacokinetics and
general toxicology. Decoy receptors will always come with additional
risks related to immunogenicity and safety, and the outstanding question
for the community is whether they provide truly unique benefits that
outweigh those risks. The advantages of soluble ACE2 are its breadth
against SARS-associated coronaviruses and its potential for alleviating
COVID-19 symptoms through intrinsic enzymatic activity. We believe these
advantages justify continued efforts towards advancing these proteins to
the clinic.

\textbf{Acknowledgements}

E.P. is supported by NIH award R01AI129719.

\textbf{Conflicts of Interest}

The University of Illinois has filed a provisional patent for engineered
decoy receptors with E.P. as an inventor. E.P. is a co-founder of
Orthogonal Biologics, Inc.

\textbf{References}

1. Adam DC, Wu P, Wong JY, Lau EHY, Tsang TK, Cauchemez S, Leung GM,
Cowling BJ. Clustering and superspreading potential of SARS-CoV-2
infections in Hong Kong. Nat Med 2020;

2. He X, Lau EHY, Wu P, Deng X, Wang J, Hao X, Lau YC, Wong JY, Guan Y,
Tan X, et al. Temporal dynamics in viral shedding and transmissibility
of COVID-19. Nat Med 2020;26:672--675.

3. Bai Y, Yao L, Wei T, Tian F, Jin D-Y, Chen L, Wang M. Presumed
Asymptomatic Carrier Transmission of COVID-19. JAMA 2020;323:1406--1407.

4. Long QX, Tang XJ, Shi QL, Li Q, Deng HJ, Yuan J, Hu JL, Xu W, Zhang
Y, Lv FJ, et al. Clinical and immunological assessment of asymptomatic
SARS-CoV-2 infections. Nat Med 2020;26:1200--1204.

5. Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W,
Lu R, et al. A Novel Coronavirus from Patients with Pneumonia in China,
2019. N Engl J Med 2020;382:727--733.

6. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, Zhang L, Fan G, Xu J, Gu
X, et al. Clinical features of patients infected with 2019 novel
coronavirus in Wuhan, China. Lancet 2020;395:497--506.

7. Zheng Z, Peng F, Xu B, Zhao J, Liu H, Peng J, Li Q, Jiang C, Zhou Y,
Liu S, et al. Risk factors of critical \& mortal COVID-19 cases: A
systematic literature review and meta-analysis. J Infect
2020;81:e16--e25.

8. Wang W, Tang J, Wei F. Updated understanding of the outbreak of 2019
novel coronavirus (2019-nCoV) in Wuhan, China. J Med Virol
2020;92:441--447.

9. Ellul MA, Benjamin L, Singh B, Lant S, Michael BD, Easton A, Kneen R,
Defres S, Sejvar J, Solomon T. Neurological associations of COVID-19.
Lancet Neurol 2020;19:767--783.

10. Becker RC. COVID-19 update: Covid-19-associated coagulopathy. J
Thromb Thrombolysis 2020;50:54--67.

11. Soy M, Keser G, Atag\selectlanguage{ngerman}ündüz P, Tabak F, Atagündüz I, Kayhan S.
Cytokine storm in COVID-19: pathogenesis and overview of
anti-inflammatory agents used in treatment. Clin Rheumatol
2020;39:2085--2094.

12. Song P, Li W, Xie J, Hou Y, You C. Cytokine storm induced by
SARS-CoV-2. Clin Chim Acta 2020;509:280--287.

13. Liu Y, Yang Y, Zhang C, Huang F, Wang F, Yuan J, Wang Z, Li J, Li J,
Feng C, et al. Clinical and biochemical indexes from 2019-nCoV infected
patients linked. Sci China Life Sci 2020;63:364--374.

14. Guo J, Huang Z, Lin L, Lv J. Coronavirus Disease 2019 (COVID-19) and
Cardiovascular Disease: A Viewpoint on the Potential Influence of
Angiotensin-Converting Enzyme Inhibitors/Angiotensin Receptor Blockers
on Onset and Severity of Severe Acute Respiratory Syndrome Coronavirus 2
Infection. J Am Heart Assoc 2020;9:e016219.

15. Roche JA, Roche R. A hypothesized role for dysregulated bradykinin
signaling in COVID-19 respiratory complications. FASEB J
2020;34:7265--7269.

16. Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Structural basis for the
recognition of SARS-CoV-2 by full-length human ACE2. Science
2020;367:1444--1448.

17. Lan J, Ge J, Yu J, Shan S, Zhou H, Fan S, Zhang Q, Shi X, Wang Q,
Zhang L, et al. Structure of the SARS-CoV-2 spike receptor-binding
domain bound to the ACE2 receptor. Nature 2020;581:215--220.

18. Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O,
Graham BS, McLellan JS. Cryo-EM structure of the 2019-nCoV spike in the
prefusion conformation. Science 2020;367:1260--1263.

19. Zhou P, Yang X Lou, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y,
Li B, Huang CL, et al. A pneumonia outbreak associated with a new
coronavirus of probable bat origin. Nature 2020;579:270--273.

20. Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor Recognition by
the Novel Coronavirus from Wuhan: an Analysis Based on Decade-Long
Structural Studies of SARS Coronavirus. J Virol 2020;94:1--9.

21. Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T,
Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, et al.
SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a
Clinically Proven Protease Inhibitor. Cell 2020;181:271-280.e8.

22. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, Somasundaran
M, Sullivan JL, Luzuriaga K, Greenough TC, et al. Angiotensin-converting
enzyme 2 is a functional receptor for the SARS coronavirus. Nature
2003;426:450--454.

23. Letko M, Marzi A, Munster V. Functional assessment of cell entry and
receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat
Microbiol 2020;5:562--569.

24. Tortorici MA, Veesler D. Chapter Four - Structural insights into
coronavirus entry. In: Rey FA, editor. Advances in Virus Research.
Academic Press; 2019. p 93--116.

25. Walls AC, Park YJ, Tortorici MA, Wall A, McGuire AT, Veesler D.
Structure, Function, and Antigenicity of the SARS-CoV-2 Spike
Glycoprotein. Cell 2020;181:281-292.e6.

26. Mille JK, Whittaker GR. Host cell entry of Middle East respiratory
syndrome coronavirus after two-step, furin-mediated activation of the
spike protein. Proc Natl Acad Sci U S A 2014;111:15214--15219.

27. Walls AC, Tortorici MA, Snijder J, Xiong X, Bosch BJ, Rey FA,
Veesler D. Tectonic conformational changes of a coronavirus spike
glycoprotein promote membrane fusion. Proc Natl Acad Sci U S A
2017;114:11157--11162.

28. Hansen J, Baum A, Pascal KE, Russo V, Giordano S, Wloga E, Fulton
BO, Yan Y, Koon K, Patel K, et al. Studies in humanized mice and
convalescent humans yield a SARS-CoV-2 antibody cocktail. Science
2020;369:1010--1014.

29. Brouwer PJM, Caniels TG, van der Straten K, Snitselaar JL, Aldon Y,
Bangaru S, Torres JL, Okba NMA, Claireaux M, Kerster G, et al. Potent
neutralizing antibodies from COVID-19 patients define multiple targets
of vulnerability. Science 2020;369:643--650.

30. Wec AZ, Wrapp D, Herbert AS, Maurer DP, Haslwanter D, Sakharkar M,
Jangra RK, Eugenia Dieterle M, Lilov A, Huang D, et al. Broad
neutralization of SARS-related viruses by human monoclonal antibodies.
Science 2020;369:731--736.

31. Wang C, Li W, Drabek D, Okba NMA, van Haperen R, Osterhaus ADME, van
Kuppeveld FJM, Haagmans BL, Grosveld F, Bosch BJ. A human monoclonal
antibody blocking SARS-CoV-2 infection. Nat Commun 2020;11:1--6.

32. Wu Y, Wang F, Shen C, Peng W, Li D, Zhao C, Li Z, Li S, Bi Y, Yang
Y, et al. A noncompeting pair of human neutralizing antibodies block
COVID-19 virus binding to its receptor ACE2. Science
2020;368:1274--1278.

33. Rogers TF, Zhao F, Huang D, Beutler N, Burns A, He W, Limbo O, Smith
C, Song G, Woehl J, et al. Isolation of potent SARS-CoV-2 neutralizing
antibodies and protection from disease in a small animal model. Science
2020;369:956-- 963.

34. Pyrc K, Dijkman R, Deng L, Jebbink MF, Ross HA, Berkhout B, van der
Hoek L. Mosaic Structure of Human Coronavirus NL63, One Thousand Years
of Evolution. J Mol Biol 2006;364:964--973.

35. Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, Liu W, Bi Y, Gao GF.
Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses.
Trends Microbiol 2016;24:490--502.

36. Baum A, Fulton BO, Wloga E, Copin R, Pascal KE, Russo V, Giordano S,
Lanza K, Negron N, Ni M, et al. Antibody cocktail to SARS-CoV-2 spike
protein prevents rapid mutational escape seen with individual
antibodies. Science 2020;369:1014-- 1018.

37. Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M,
Leopoldi A, Garreta E, Hurtado del Pozo C, Prosper F, et al. Inhibition
of SARS-CoV-2 Infections in Engineered Human Tissues Using
Clinical-Grade Soluble Human ACE2. Cell 2020;181:905-913.e7.

38. Lei C, Qian K, Li T, Zhang S, Fu W, Ding M, Hu S. Neutralization of
SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig. Nat Commun
2020;11:1--5.

39. Hofmann H, Geier M, Marzi A, Krumbiegel M, Peipp M, Fey GH, Gramberg
T, Pöhlmann S. Susceptibility to SARS coronavirus S protein-driven
infection correlates with expression of angiotensin converting enzyme 2
and infection can be blocked by soluble receptor. Biochem Biophys Res
Commun 2004;319:1216--1221.

40. Chan KK, Tan TJC, Narayanan KK, Procko E. An engineered decoy
receptor for SARS-CoV-2 broadly binds protein S sequence variants.
bioRxiv 2020;10.18.344622.

41. Usmani SS, Bedi G, Samuel JS, Singh S, Kalra S, Kumar P, Ahuja AA,
Sharma M, Gautam A, Raghava GPS. THPdb: Database of FDA-approved peptide
and protein therapeutics. PLoS One 2017;12:1--12.

42. Zoufaly A, Poglitsch M, Aberle JH, Hoepler W, Seitz T, Traugott M,
Grieb A, Pawelka E, Laferl H, Wenisch C, et al. Human recombinant
soluble ACE2 in severe COVID-19. Lancet Respir Med 2020;2600:1--5.

43. Chan KK, Dorosky D, Sharma P, Abbasi SA, Dye JM, Kranz DM, Herbert
AS, Procko E. Engineering human ACE2 to optimize binding to the spike
protein of SARS coronavirus 2. Science 2020;369:1261--1265.

44. Procko E. The sequence of human ACE2 is suboptimal for binding the S
spike protein of SARS coronavirus 2. bioRxiv 2020;03.16.994236.

45. Procko E. Deep mutagenesis in the study of COVID-19: a technical
overview for the proteomics community. Expert Rev Proteomics 2020;1--6.

46. Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS.
Engineering and characterization of a superfolder green fluorescent
protein. Nat Biotechnol 2006;24:79--88.

47. Heinzelman P, Romero PA. Discovery of human ACE2 variants with
altered recognition by the SARS-CoV-2 spike protein. bioRxiv
2020;09.17.301861.

48. Rushworth CA, Guy JL, Turner AJ. Residues affecting the chloride
regulation and substrate selectivity of the angiotensin-converting
enzymes (ACE and ACE2) identified by site-directed mutagenesis. FEBS J
2008;275:6033--6042.

49. Higuchi Y, Suzuki T, Arimori T, Ikemura N, Kirita Y, Ohgitani E,
Mazda O, Motooka D, Nakamura S, Matsuura Y, et al. High affinity
modified ACE2 receptors prevent SARS-CoV-2 infection. bioRxiv
2020;09.16.299891.

50. Glasgow A, Glasgow J, Limonta D, Solomon P, Lui I, Zhang Y, Nix MA,
Rettko NJ, Zha S, Yamin R, et al. Engineered ACE2 receptor traps
potently neutralize SARS-CoV-2. Proc Natl Acad Sci 2020;202016093.

51. Moore MJ, Dorfman T, Li W, Wong SK, Li Y, Kuhn JH, Coderre J,
Vasilieva N, Han Z, Greenough TC, et al. Retroviruses Pseudotyped with
the Severe Acute Respiratory Syndrome Coronavirus Spike Protein
Efficiently Infect Cells Expressing Angiotensin-Converting Enzyme 2. J
Virol 2004;78:10628--10635.

52. Li Y, Wang H, Tang X, Fang S, Ma D, Du C, Wang Y, Pan H, Yao W,
Zhang R, et al. SARS-CoV-2 and three related coronaviruses utilize
multiple ACE2 orthologs and are potently blocked by an improved ACE2-Ig.
J Virol 2020;

53. Iwanaga N, Cooper L, Rong L, Beddingfield B, Crabtree J, Tripp A,
Qin X, Kolls JK. Novel ACE2-IgG1 fusions with improved in vitro and in
vivo activity against SARS-CoV2. bioRxiv 2020;06.15.152157.

54. Lu J, Sun PD. High affinity binding of SARS-CoV-2 spike protein
enhances ACE2 carboxypeptidase activity. J Biol Chem 2020;RA120:015303.

55. Wysocki J, Ye M, Rodriguez E, González-Pacheco FR, Barrios C, Evora
K, Schuster M, Loibner H, Brosnihan KB, Ferrario CM, et al. Targeting
the degradation of angiotensin II with recombinant
angiotensin-converting enzyme 2: Prevention of angiotensin II-dependent
hypertension. Hypertension 2010;55:90--98.

56. Haschke M, Schuster M, Poglitsch M, Loibner H, Salzberg M,
Bruggisser M, Penninger J, Krähenbühl S. Pharmacokinetics and
pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in
healthy human subjects. Clin Pharmacokinet 2013;52:783--792.

57. Strohl WR. Fusion Proteins for Half-Life Extension of Biologics as a
Strategy to Make Biobetters. BioDrugs 2015;29:215--239.

58. Crackower MA, Sarao R, Oliveira-dos-Santos AJ, Da Costa J, Zhang L.
Angiotensin-converting enzyme 2 is an essential regulator of heart
function. Nature 2002;417:822--828.

59. Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R, Wada
T, Leong-Poi H, et al. Angiotensin-converting enzyme 2 protects from
severe acute lung failure. Nature 2005;436:112--116.

60. Zou Z, Yan Y, Shu Y, Gao R, Sun Y, Li X, Ju X, Liang Z, Liu Q, Zhao
Y, et al. Angiotensin-converting enzyme 2 protects from lethal avian
influenza A H5N1 infections. Nat Commun 2014;5:1--7.

61. Treml B, Neu N, Kleinsasser A, Gritsch C, Finsterwalder T, Geiger R,
Schuster M, Janzek E, Loibner H, Penninger J, et al. Recombinant
angiotensin-converting enzyme 2 improves pulmonary blood flow and
oxygenation in lipopolysaccharide-induced lung injury in piglets. Crit
Care Med 2010;38.

62. Johnson JA, West J, Maynard KB, Hemnes AR. ACE2 improves right
ventricular function in a pressure overload model. PLoS One 2011;6.

63. Liu P, Wysocki J, Souma T, Ye M, Ramirez V, Zhou B, Wilsbacher LD,
Quaggin SE, Batlle D, Jin J. Novel ACE2-Fc chimeric fusion provides
long-lasting hypertension control and organ protection in mouse models
of systemic renin angiotensin system activation. Kidney Int
2018;94:114--125.

64. Khan A, Benthin C, Zeno B, Albertson TE, Boyd J, Christie JD, Hall
R, Poirier G, Ronco JJ, Tidswell M, et al. A pilot clinical trial of
recombinant human angiotensin-converting enzyme 2 in acute respiratory
distress syndrome. Crit Care 2017;21:1--9.

65. Saunders KO. Conceptual Approaches to Modulating Antibody Effector
Functions and Circulation Half-Life. Front Immunol 2019;10:1296.

66. Park J, Gill KS, Aghajani AA, Heredia JD, Choi H, Oberstein A,
Procko E. Engineered receptors for human cytomegalovirus that are
orthogonal to normal human biology. PLOS Pathog 2020;16:e1008647.

67. Kuba K, Imai Y, Ohto-Nakanishi T, Penninger JM. Trilogy of ACE2: A
peptidase in the renin--angiotensin system, a SARS receptor, and a
partner for amino acid transporters. Pharmacol Ther 2010;128:119--128.

68. Gheblawi M, Wang K, Viveiros A, Nguyen Q, Zhong J-C, Turner AJ,
Raizada MK, Grant MB, Oudit GY. Angiotensin-Converting Enzyme 2:
SARS-CoV-2 Receptor and Regulator of the Renin-Angiotensin System:
Celebrating the 20th Anniversary of the Discovery of ACE2. Circ Res
2020;126:1456--1474.

69. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, Turner AJ. A
human homolog of angiotensin-converting enzyme. Cloning and functional
expression as a captopril-insensitive carboxypeptidase. J Biol Chem
2000;275:33238--33243.

70. Turner AJ, Tipnis SR, Guy JL, Rice G, Hooper NM. ACEH/ACE2 is a
novel mammalian metallocarboxypeptidase and a homologue of
angiotensin-converting enzyme insensitive to ACE inhibitors. Can J
Physiol Pharmacol 2002;80:346--353.

71. Vickers C, Hales P, Kaushik V, Dick L, Gavin J, Tang J, Godbout K,
Parsons T, Baronas E, Hsieh F, et al. Hydrolysis of biological peptides
by human angiotensin-converting enzyme-related carboxypeptidase. J Biol
Chem 2002;277:14838--14843.

72. Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N,
Donovan M, Woolf B, Robison K, Jeyaseelan R, et al. A novel
angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts
angiotensin I to angiotensin 1-9. Circ Res 2000;87:E1-9.

73. Dasgupta C, Zhang L. Angiotensin II receptors and drug discovery in
cardiovascular disease. Drug Discov Today 2011;16:22--34.

74. Garvin MR, Alvarez C, Miller JI, Prates ET, Walker AM, Amos BK, Mast
AE, Justice A, Aronow B, Jacobson D. A mechanistic model and therapeutic
interventions for COVID-19 involving a RAS-mediated bradykinin storm.
Elife 2020;9:e59177.

75. Chen P, Nirula A, Heller B, Gottlieb RL, Boscia J, Morris J, Huhn G,
Cardona J, Mocherla B, Stosor V, et al. SARS-CoV-2 Neutralizing Antibody
LY-CoV555 in Outpatients with Covid-19. N Engl J Med 2020;

76. Liu P, Xie X, Gao L, Jin J. Designed Variants of Recombinant ACE2-Fc
that Decouple Anti-SARS-CoV-2 Activities from Unwanted Cardiovascular
Effects. bioRxiv 2020;08.13.248351.

77. Huang Y, Yang C, Xu X, Xu W, Liu S. Structural and functional
properties of SARS-CoV-2 spike protein: potential antivirus drug
development for COVID-19. Acta Pharmacol Sin 2020;41:1141--1149.

78. Ke Z, Oton J, Qu K, Cortese M, Zila V, McKeane L, Nakane T, Zivanov
J, Neufeldt CJ, Cerikan B, et al. Structures and distributions of
SARS-CoV-2 spike proteins on intact virions. Nature 2020;

79. Turo\selectlanguage{polish}ň\selectlanguage{english}ov\selectlanguage{ngerman}á B, Sikora M, Schürmann C, Hagen WJH, Welsch S, Blanc FEC,
von Bülow S, Gecht M, Bagola K, Hörner C, et al. In situ structural
analysis of SARS-CoV-2 spike reveals flexibility mediated by three
hinges. Science 2020;370:203-- 208.

80. Yao H, Song Y, Chen Y, Wu N, Xu J, Sun C, Zhang J, Weng T, Zhang Z,
Wu Z, et al. Molecular Architecture of the SARS-CoV-2 Virus. Cell
2020;183:730-738.e13.

81. Lui I, Zhou XX, Lim SA, Elledge SK, Solomon P, Rettko NJ, Zha BS,
Kirkemo LL, Gramespacher JA, Liu J, et al. Trimeric SARS-CoV-2 Spike
interacts with dimeric ACE2 with limited intra-Spike avidity. bioRxiv
2020;05.21.109157.

82. Guo L, Bi W, Wang X, Xu W, Yan R, Zhang Y, Zhao K, Li Y, Zhang M,
Bao X, et al. Engineered Trimeric ACE2 Binds and Locks ``Three-up''
Spike Protein to Potently Inhibit SARS-CoVs and Mutants. bioRxiv
2020;2020.08.31.274704.

83. Xiao T, Lu J, Zhang J, Johnson RI, McKay LGA, Storm N, Lavine CL,
Peng H, Cai Y, Rits-Volloch S, et al. A trimeric human
angiotensin-converting enzyme 2 as an anti-SARS-CoV-2 agent in vitro.
bioRxiv 2020;09.18.301952.

84. Miao X, Luo Y, Huang X, Lee SMY, Yuan Z, Tang Y, Chen L, Wang C, Wu
F, Xu Y, et al. A novel biparatopic hybrid antibody-ACE2 fusion that
blocks SARS-CoV-2 infection: implications for therapy. MAbs 2020;12.

85. Wang C, Wang S, 1\# C, Zhao J, Han S, Zhao G, Kang J, Liu Y, Wang L,
Wang X, et al. Membrane Nanoparticles Derived from ACE2-rich Cells Block
SARS-CoV-2 Infection. bioRxiv 2020;08.12.247338.

86. Cocozza F, Piovesana E, Névo N, Lahaye X, Buchrieser J, Schwartz O,
Manel N, Tkach M, Théry C, Martin-Jaular L. Extracellular vesicles
containing ACE2 efficiently prevent infection by SARS-CoV-2 Spike
protein-containing virus. bioRxiv 2020;07.08.193672.

87. Rao L, Xia S, Xu W, Tian R, Yu G, Gu C, Pan P, Meng Q-F, Cai X, Qu
D, et al. Decoy nanoparticles protect against COVID-19 by concurrently
adsorbing viruses and inflammatory cytokines. Proc Natl Acad Sci
2020;2:202014352.

\textbf{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).

\textbf{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.\textbf{(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.\textbf{(C)} Structure (PDB 6M17) of RBD (green) bound to ACE2
(pale blue). Common mutations in high affinity engineered decoys are
indicated in parentheses. \textbf{(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\textsuperscript{43}.

\textbf{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 Fc\selectlanguage{greek}γ\selectlanguage{english}Rs 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. \textbf{(B)} The conversion of angiotensin peptide hormones
by renin, ACE1 and ACE2.

\textbf{Figure 4. 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.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure1/Figure1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure2/Figure2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure3/Figure3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure4/Figure4}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

