key: cord-0909104-dkgi0eqv
authors: Svilenov, Hristo L.; Sacherl, Julia; Reiter, Alwin; Wolff, Lisa S.; Cheng, Cho-Chin; Stern, Marcel; Grass, Vincent; Feuerherd, Martin; Wachs, Frank-Peter; Simonavicius, Nicole; Pippig, Susanne; Wolschin, Florian; Keppler, Oliver T.; Buchner, Johannes; Brockmeyer, Carsten; Protzer, Ulrike
title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein
date: 2021-11-10
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2021.105197
sha: b9d127a3bd4c1979a4c079fb2ecfa9c4d6cbd4e5
doc_id: 909104
cord_uid: dkgi0eqv

SARS-CoV-2 enters host cells after binding through its spike glycoprotein to the angiotensin-converting enzyme 2 (ACE2) receptor. Soluble ACE2 ectodomains bind and neutralize the virus, yet their short in vivo half-live limits their therapeutic use. This limitation can be overcome by fusing the fragment crystallizable (Fc) part of human immunoglobulin G (IgG) to the ACE2 ectodomain, but this bears the risk of Fc-receptor activation and antibody-dependent cellular cytotoxicity. Here, we describe optimized ACE2-IgG4-Fc fusion constructs that avoid Fc-receptor activation, preserve the desired ACE-2 enzymatic activity and show promising pharmaceutical properties. The engineered ACE2-IgG4-Fc fusion proteins neutralize the original SARS-CoV, pandemic SARS-CoV-2 as well as the rapidly spreading SARS-CoV-2 alpha, beta and delta variants-of-concern. Importantly, these variants-of-concern are inhibited at picomolar concentrations proving that ACE-2-IgG4 maintains – in contrast to therapeutic antibodies - its full antiviral potential. Thus, ACE2-IgG4-Fc fusion proteins are promising candidate anti-antivirals to combat the current and future pandemics.

Angiotensin-converting enzyme 2 (ACE2) serves as a common entry receptor for the human coronavirus (CoV)-NL63, the original SARS-CoV from 2003 and the novel, pandemic SARS-CoV-2 into their host cells by binding the spike protein on the virus surface Hofmann et al., 2005; Wrapp et al., 2020) . Membrane fusion of SARS-CoV-2 is enabled by proteolytic activation of the spike protein by cell surface protease TMPRSS2 . In addition, neuropilin-1 has recently been identified to bind a furin-cleaved spike protein and by this to significantly potentiate SARS-CoV-2 infectivity (Cantuti-Castelvetri et al., 2020) .

ACE2 is expressed on the plasma membrane of epithelial cells in the respiratory tract and lung (Wrapp et al., 2020) , but also in other tissues like intestine, testes, liver, kidney, brain, and in the cardiovascular system (Crackower et al., 2002; Ding et al., 2004; Hamming et al., 2004) .

ACE2 is an 805 amino acid type-I transmembrane protein consisting of extracellular, transmembrane and cytosolic domains (Jiang et al., 2014) . The extracellular domain is a zinc metalloprotease, which enzymatically functions as a carboxypeptidase (Donoghue et al., 2000; Tipnis et al., 2000) suppressing the renin-angiotensin-aldosterone system (RAAS) by cleaving angiotensin II into heptapeptide angiotensin-(1-7) and cleaving other vasoactive peptides (Lu et al., 2016; Santos et al., 2018) . Angiotensin-(1-7) lowers the diastolic blood pressure, has antiinflammatory, anti-proliferative and anti-fibrotic effects (Burrell et al., 2004) and thereby protects lung, heart, kidney and other organs from injury (Crackower et al., 2002; Ding et al., 2004; Hamming et al., 2004) . The potential contribution of angiotensin II to the COVID-19 pathophysiology has been indicated by reports that angiotensin II levels in plasma samples from COVID-19 patients were markedly elevated and correlated with viral load and severity of the disease (Liu et al., 2020a; Wu et al., 2020) .

ACE2-Fc fusion proteins, composed of an IgG Fc domain fused to the extracellular domain of ACE2, have been suggested as a high-priority treatment option for COVID-19 (Batlle et al., 2020;  J o u r n a l P r e -p r o o f Kruse, 2020) . In addition to neutralizing SARS-CoV and SARS-CoV-2, the ACE2's enzymatic activity affecting the RAAS provides a second mode of action, potentially alleviating the pathophysiology of acute respiratory distress syndrome. Therapeutic use of a soluble human recombinant ACE2 dimer (APN01) with a half-life of 10 hours is currently investigated in patients with COVID-19 (Haschke et al., 2013; Khan et al., 2017; Zoufaly et al., 2020) . Strong in vitro SARS-CoV-2 neutralizing activity has been described for sequence variants of ACE2-IgG1-Fc fusion proteins (Glasgow et al., 2020; Higuchi Y, 2021; Huang et al., 2020; Iwanaga et al., 2020; Lei et al., 2020; Liu et al., 2020b; Lui et al., 2020) , and ACE2-IgG1-Fc (HLX71) has entered phase 1 clinical studies.

A theoretical concern at the beginning of the pandemic was antibody-mediated enhancement (ADE) of SARS-CoV-2 infection, or disease-enhancing pathophysiology of sub-neutralizing or cross-reactive non-neutralizing antibodies by Fc-mediated complement activation or antibodydependent cellular cytotoxicity (Bournazos et al., 2020) . For the SARS-CoV-2 relative Middle East respiratory syndrome (MERS)-CoV, binding of Fc receptor gamma III (CD16) has led to infection of CD16 positive cells (Jafarzadeh et al., 2020; Manickam et al., 2020) . While IgG1-Fc strongly binds to CD16 and inducing pronounced cytotoxicity, Fc-related effector functions are minimal for IgG4-Fc (de Taeye et al., 2020) . In this regard, the IgG4-Fc fragment appeared to be a preferred fusion partner for ACE2. However, it is well known that naturally occurring IgG4 antibodies are less stable than IgG1 variants due to the formation of half antibodies, which limits their use in pharmaceutical preparations (Aalberse and Schuurman, 2002; Correia, 2010; Dumet et al., 2019; Handlogten et al., 2020) . To generate a stable ACE2-IgG4-Fc fusion protein, we have therefore chosen the immunoglobulin Fc region of an IgG4/kappa isotype with an S228P sequence alteration in the hinge region (Aalberse and Schuurman, 2002) . For the ACE2 domain, two different truncations (Q18-G732 and Q18-S740) were used, and point mutations were introduced in the ACE2 domain to abrogate its enzymatic activity.

J o u r n a l P r e -p r o o f

Construct design ACE2 amino acid sequence modifications were designed by computer-aided modelling. ACE2 ectodomains of different length, Q18-G732 and Q18-S740, with or without mutation of the catalytic site (wild type or H374N/H378N mutant) (Moore et al., 2004) were combined with the Fc fragment of IgG4 bearing a stabilizing S228P mutation in the hinge region (Aalberse and Schuurman, 2002) .

For comparison, the same ACE2 sequence variants were fused to the Fc fragment of IgG1 with a truncated hinge region (DKTHTCPPCPA).

Plasmids encoding the Fc fusion proteins were generated at ThermoFisher. Genes of interest were subcloned into pcDNA3.1 Zeocin expression plasmids (Invitrogen V860-20, Life

Technologies, Carlsbad, CA, USA) with an elongated CMV promoter using HindIII/XhoI restriction sites. Following amplification in Escherichia coli, expression plasmids were isolated and analyzed by restriction analysis as well as DNA sequencing.

Using the FreeStyle 293 Expression System (Thermo Fisher Scientific, Waltham, MA, USA), the different ACE2-Fc fusion proteins were transiently expressed in 3 x 240 mL culture media. On day six, samples were analyzed for cell viability as well as cell density and supernatants were harvested by centrifugation followed by sterile filtration (Walls et al., 2020) . The material was either stored at -80°C until purification or subjected directly to purification. Small samples were taken from the pools to determine expression yields by bio-layer interferometry (BLI).

Purification of the fusion proteins secreted into the culture medium was performed by protein A column chromatography followed by preparative Size Exclusion Chromatography (SEC). For J o u r n a l P r e -p r o o f protein A purification, after loading the sample, the Amsphere A3 column (JSR Life Sciences, Sunnyvale, CA, USA) was washed and the ACE2-Fc fusion proteins were eluted using 40 mM sodium acetate buffer, pH 3.0. Following elution, samples were first neutralized to pH 7.5 using 1 M Tris, pH 9.0, subsequently diluted 1:1 with 50 mM Tris, pH 7.5, 300 mM NaCl and concentrated to 10 mg/mL using spin filters. Concentrated proteins were further purified with a Superdex 200 increase (GE Healthcare-Cytiva, Chicago, IL, USA) column equilibrated with 50 mM Tris, pH 7.5, 150 mM NaCl. The main peak was pooled, the protein concentration was determined by slope spectrometry (Lehr et al., 2015) and adjusted to 1 mg/mL. The protein solution was passed through a sterilizing filter and stored at 4°C until further usage.

Size exclusion chromatography with multi-angle light scattering (SEC-MALS) A Shimadzu HPLC system with two concentration detectors (UV and refractive index) and a HELEOS II MALS detector were used for the measurements. The flow rate was 1 mL/min and the running buffer was 50 mM Tris, pH 7.5 and 150 mM NaCl. 50 µg of protein was injected on a Superdex 200 Increase 10/300 GL column (Cytiva). The chromatograms were evaluated with the Astra software.

2.6. Circular dichroism (CD)

All CD measurements were performed with a Jasco J-1500 spectropolarimeter at 20°C. The sample buffer consisted of 50 mM Tris, pH 7.5 and 150 mM NaCl. The Far-UV CD spectra were obtained in a 1 mm quartz cuvette using a protein concentration of 0.1 mg/mL. The Near-UV CD spectra were measured in a 5 mm quartz cuvette using a protein concentration of 1 mg/mL. J o u r n a l P r e -p r o o f 2.10. SARS-CoV-2 Spike S1 Inhibition ELISA Inhibition of binding of SARS-CoV-2 spike S1 protein to ACE2 was tested using the ACE2:SARS-CoV-2 Spike S1 Inhibitor Screening Assay Kit (BPS Bioscience, San Diego, CA, USA; Cat.No.

79945) according to the manufacturer's instructions with an adapted neutralization procedure.

Briefly, biotinylated SARS-CoV-2 Spike S1 protein (25 nM) was incubated with serial dilutions of the ACE2-Fc fusion proteins in a 96-well neutralization plate at room temperature (RT) for one hour with slow shaking (= neutralization mix).

ACE2 protein was added to a nickel-coated 96-well plate at a concentration of 1 μg/mL and incubated at RT for one hour with slow shaking. Following a washing step to remove unbound ACE2, the plates were blocked at RT for 10 min with slow shaking. Subsequently, the neutralization mix was transferred to the ACE2 coated plate and the plate was incubated at RT for one hour with slow shaking. Following a 10 min blocking step, the plate was incubated with

Streptavidin-HRP at RT for one hour with slow shaking. Following a washing and a 10 min blocking step, the HRP substrate was added and the plate was analyzed on a chemiluminescence reader. Shanghai (Wolf et al., 2020) . SARS-CoV-2-April was isolated during the first eminent wave of the pandemic in Europe in April 2020 from a patient in Munich, Germany. Both virus isolates as well as a control isolate from the early "Webasto" cluster outbreak contain the S1 D614G mutation showing significantly higher infectious titers in vitro (Korber et al., 2020) . Briefly, HepG2 or Vero E6 cells were plated in a 12-well plate at 5E05 cells/well in DMEM medium supplemented with 5% FCS, 1% P/S, 200 mmol/L L-glutamine, 1% MEM-NEAA, 1% sodiumpyruvate and incubated overnight at 37°C and 5% CO2. Cells were infected with serial dilution of virus sample in cell culture medium at 37°C for one hour. After discarding the supernatant, 1 mL of 5% carboxymethylcellulose diluted in Minimum Essential Media was added per well and the plate was incubated at 37°C until obvious plaques appeared. After removing the supernatant, cells were fixed with 10% paraformaldehyde at RT for 30 min. Next, a washing step with PBS was performed, followed by the addition of 1% crystal violet (diluted in 20% methanol and water).

Following an incubation time of 15 min at RT, the solution was washed away with PBS and the J o u r n a l P r e -p r o o f plate was dried. The viral titer (PFU/mL) of the sample was determined by counting the average number of plaques for a dilution and the inverse of the total dilution factor.

Vero-E6 cells were plated in a 96-well plate at 1.4E04 cells/well in DMEM medium (Gibco) supplemented with 5% FCS, 1% P/S, 200 mmol/L L-glutamine, 1% MEM-NEAA, 1% sodiumpyruvate (all from Gibco) and incubated overnight at 37°C and 5% CO2. Serial dilutions of ACE2-Fc fusion proteins and SARS-CoV-2-GFP were mixed in fresh media and pre-incubated at 37°C 

First, we wanted to create ACE2-Fc molecules with different biological properties using the PyMOL Molecular Graphics System (version 2.3.3., Schrödinger, LLC., 2020). ACE2-IgG4-Fc and ACE2-IgG1-Fc fusion proteins were designed based on crystal and EM structures of the ACE2 extracellular domain, the SARS-CoV-2 spike (S) protein and its receptor-binding domain (RBD), as well as the IgG4-Fc and IgG1-Fc domains (Figure 1a and b ) (Lan et al., 2020; Scapin et al., 2015; Wrapp et al., 2020; Yan et al., 2020) . Details of the ACE2 sequences fused to the Fc fragments of IgG4 and IgG1 are shown in Figure 1c .

The expression yields of all constructs were in a similar range, although slightly higher for Q18-G732 ACE2-Fc fusion proteins (Supplementary Table 1 J o u r n a l P r e -p r o o f

To study the secondary structure to the ACE2-Fc fusion construct, we used protein circular dichroism (CD). The Far-UV (200-260 nm) CD spectra of the fusion proteins could be superimposed indicating that the secondary structures are preserved among all constructs, regardless of the sequence variations (Figure 2a) . The same held true for the Near-UV (250-350 nm) CD spectra, which indicated that the overall tertiary structure is also highly similar in all ACE2-Fc proteins investigated (Figure 2a) . 

Surface plasmon resonance (SPR) allowed us to determine the binding affinity of our ACE2-Fc constructs to the RBD of the spike protein of SARS-CoV-2 that was recombinantly expressed and immobilized. The ACE2-Fc fusion proteins bound in a concentration-dependent manner to the viral protein domain, while an unrelated Fc fusion protein (aflibercept) used as a control showed no interaction with the ligand (Figure 3a) . The binding constants revealed that all constructs analyzed bind to the immobilized SARS-CoV-2 RBD with an equilibrium dissociation constant (KD) of around 4 nM (Figure 3b ). This indicated that the structural variations do not influence the interaction between the ACE2-Fc fusion proteins and the RBD of SARS-CoV-2.

Neutralizing activities of the ACE2-Fc fusion proteins against the SARS-CoV-2 spike protein were tested in a competition enzyme-linked immunoassay (ELISA). All ACE2-Fc constructs tested potently inhibited the binding of spike S1 protein of SARS-CoV-2 to ACE2 (Figure 3c) . Consistent with their affinities to the SARS-CoV-2 RBD, there were no significant differences between the different fusion proteins. The half-maximal inhibitory concentrations (IC50 values) for the SARS-CoV-2 spike S1 protein ranged from 2.5 to 3.5 nM.

. c ACE2-Fc fusion proteins were pre-incubated with the SARS-CoV-2 spike S1 protein and tested in a competition ELISA for their ability to neutralize S1 binding to immobilized ACE2 protein. Potent inhibition of SARS-CoV-2 spike S1 protein by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right). Data are represented as means ± SD of at least two independent experiments.

The Fc-part can be important for the interaction of the molecules with Fc-receptors. We therefore used SPR to test the interaction of two ACE2-IgG4-Fc and two ACE2-IgG1-Fc proteins with FcRI and FcRIIIa. The ACE2-IgG4-Fc fusion proteins showed slightly lower affinity to FcRI when compared to the IgG1 counterparts ( Mean ± SD of duplicate measurements.

We next wanted to test whether the competition of our ACE2-Fc fusion constructs with binding of the SARS-CoV-2 via S1 to its receptor ACE2 translates into neutralization of infectious virus. 

To determine whether the ACE2-Fc constructs neutralize different isolates of SARS-CoV-2, we compared the neutralization capacity of each of the eight ACE2-Fc fusion constructs against a range of primary isolates from patients. Hereby, we also included the original SARS-CoV isolate from 2003 (Drosten et al., 2003) Second, to test the ACE2-Fc constructs for their ability to neutralize SARS-CoV-2, we evaluated the antiviral activity against SARS-CoV-2-Jan isolated from the earliest documented COVID-19 patient in Germany (Bohmer et al., 2020; Rothe et al., 2020) , which was directly connected to the initial outbreak in Wuhan, China. All ACE2-Fc fusion proteins displayed strong neutralizing potential against SARS-CoV-2-Jan (Figure 4b) with IC50 values in the range of 7-11 nM.

Third, the ACE2-Fc fusion protein variants were also compared for their ability to neutralize a second SARS-CoV-2 isolate, SARS-CoV-2-April, which was isolated when the virus was massively spreading in Europe. SARS-CoV-2-April displayed a different plaque-forming phenotype than SARS-CoV-2-Jan (Supplementary Figure 1) . All ACE2-Fc constructs displayed significantly increased neutralizing potential against SARS-CoV-2-April (Figure 4b) with IC50 in the picomolar range. The comparison of IC50 values showed an increasing neutralizing potential of ACE2-Fc fusion proteins, the more the SARS coronavirus evolved to allow for more efficient spread in the community.

J o u r n a l P r e -p r o o f

To confirm our results obtained in VeroE6 cells, we determine whether the ACE2-Fc fusion proteins can prevent cell toxicity resulting from infection with SARS-CoV-2 VoC in human alveolar basal epithelium-derived A549-hACE2 cells. A549-hACE2 cells were challenged with the different ACE2-Fc fusion protein-pre-treated VoCs. Because of their expected favorable in vivo features, we chose the two IgG4-based ACE2-Fc fusion proteins, construct 1 and construct 3 (Figure 5) .

Both ACE2-IgG4-Fc constructs entirely prevented SARS-CoV-2-induced cytotoxicity and, therefore, potently neutralized SARS-CoV-2-Jan ( Figure 5 ) and SARS-CoV-2-April ( Figure 5) with IC50 values in the range of 1-7 nM confirming previous results (Figure 4b ).

In 

In this study, we designed, expressed and evaluated different ACE2-Fc fusion proteins with favorable biochemical features and demonstrate that they elicit a broad antiviral activity in the nano-molar or even picomolar range against human pathogenic beta-coronaviruses including SARS-CoV-2 VoCs as well as the 2003 SARS-CoV. Importantly, the more the SARS-CoV-2 spike protein evolved and diversified in the pandemic, and the more problematic immune escape became, the more efficient these virus isolates were inhibited by our ACE2-Fc fusion proteins. in Germany, and thus is closely related to the original Wuhan strain (Bohmer et al., 2020; Rothe et al., 2020; Wolf et al., 2020) , was inhibited with IC50 values of around 10 nM. Infection by the SARS-CoV-2-April variant predominantly circulating worldwide was prevented even more efficiently. Most importantly, our ACE2-IgG4-Fc constructs prevented infection and cell toxicity of the most infectious SARS-CoV-2 variant known so far, the alpha strain, and the variants of highest concern world-wide, the beta and delta variants reaching picomolar IC50. This demonstrates that, in contrast to therapeutic antibodies targeting the viral spike protein, the virus cannot escape neutralization by or ACE2-IgG4-Fc fusion construct.

Surveillance of SARS-CoV-2 variants emerging over time identified genomic regions with increasing genetic variation (Islam et al., 2020; Khan et al., 2020; Mercatelli and Giorgi, 2020) . A D614G substitution in the C-terminal region stabilizing the spike protein is associated with an improved ability of the virus to bind its receptor, ACE2, and rapidly became the most prevalent variant worldwide (Fernandez, 2020; Korber et al., 2020) . Over time, novel and even more infectious SARS-CoV-2 strains emerged, the alpha variant being the prime example (Santos and Passos, 2021) . Our results indicate that both the ACE2-IgG4-Fc fusion proteins and the ACE2-J o u r n a l P r e -p r o o f IGg1-Fc fusion proteins neutralize these pandemic SARS-CoV-2 variants even more potently than the early SARS-CoV-2, which originated from Wuhan. The lower efficacy against the original SARS-CoV from 2003 is in line with the fact that the affinity of the SARS-CoV-2 spike protein for ACE2 is significantly higher than the affinity of the SARS-CoV spike protein (Wrapp et al., 2020) .

It, however, also indicates, that the VoCs evolving because they can bind more efficiently to ACE2

and therefore become more infectious can be targeted with our ACE2-IgG4 fusion protein even more efficiently. This helps to prepare for new potentially pandemic viruses stemming from animal reservoirs, as it happened recently with "cluster 5", a SARS-CoV-2 variant with a combination of mutations still investigated for their impact on disease severity and resistance to vaccination and therapeutic antibodies (Mallapaty, 2020; Oude Munnink et al., 2021) .

Our findings are highly relevant in the context of the emerging SARS-CoV-2 variants that at least partially escape neutralization by therapeutic antibodies (Hu et al., 2021; Wang et al., 2021) . The use of one of the first antibodies used in COVID-19 therapy, bamlanivimab, was even halted by the US-FDA only four months after it had gained authorization for emergency use. The FDA is now recommending to develop and use antibodies only for combination therapies targeting different epitopes due to viral resistance concerns. However, it is probably only a matter of time until we see SARS-CoV-2 variants emerging that escape multiple therapeutic antibodies. Unlike antibodies, our ACE2-IgG4-Fc fusion protein will retain its potent virus neutralization efficiency as it is vital for the virus to retain its receptor binding capacity. Therefore, coronavirus variants that mutate to escape ACE2-Fc-binding will lose fitness and become less infectious.

In addition to the alpha VoC, the emergence of SARS-CoV-2 VoCs which are able to at least partially escape immunity raises additional concerns, namely those which carry a mutation of amino acid 484 such as the beta, the gamma and the most recent delta variant detected in India.

SARS-CoV-2 VoC alpha has been shown to be refractory to neutralization by most monoclonal antibodies against the N-terminal domain of the spike protein and is relatively resistant to a few J o u r n a l P r e -p r o o f monoclonal antibodies against the receptor-binding domain while it is still sensitive to plasma from individuals who have recovered from COVID-19 or sera from individuals who have been vaccinated against SARS-CoV-2. The beta and delta VoC are not only refractory to neutralization by most monoclonal antibodies directed against the N-terminal domain of the spike protein but also by multiple individual monoclonal antibodies against the receptor-binding motif. In addition, both variants show a worrisome escape from neutralization by convalescent plasma and sera from individuals who have been vaccinated (Garcia-Beltran et al., 2021; Planas et al., 2021; Wang et al., 2021) . Our data strongly indicate that all these variants can still be targeted by the engineered ACE2-IgG4 fusion constructs.

ACE2 plays a central role in the homeostatic control of cardio-renal actions and has been shown to protect against severe acute lung injury and acute angiotensin II-induced hypertension (Tikellis and Thomas, 2012; Wysocki et al., 2010) . A soluble dimer (APN019) has been safely tested in a clinical phase I study in healthy volunteers and in a phase II study in patients with an acute respiratory distress syndrome (Haschke et al., 2013; Khan et al., 2017) . It is currently being tested for its therapeutic effect in a phase II study in COVID-19 patients (Zoufaly et al., 2020) . We used the full length ACE2 ectodomain sequence Q18-S740 and a shortened version comprising Q18-G732. Most importantly, binding to the SARS-CoV-2 viral spike protein to both variants was comparable with a KD of 4 nM.

Pharmacokinetic studies in mice and humans revealed that recombinant human ACE2 exhibits fast clearance rates resulting in a short half-life of only a few hours (Haschke et al., 2013; Wysocki et al., 2010; Zoufaly et al., 2020) . When the extracellular domain of murine ACE2 was fused to an to IgGs (Bernardi et al., 2020) . Functional variants of ACE2-IgG1-Fc fusion proteins have been described in the literature (Glasgow et al., 2020; Huang et al., 2020; Iwanaga et al., 2020; Lei et al., 2020; Liu et al., 2020b; Lui et al., 2020) , and a first fusion protein designed with an IgG1 Fc portion to prolong the circulating half-life is currently in a phase II clinical study by Hengenix

Biotech Inc. In our study, novel ACE2-Fc fusion proteins were designed in which the ACE2 domain is fused to the Fc fragment of human IgG4 (ACE2-IgG4-Fc) containing a stabilizing S228P mutation in the hinge region. Although ACE2 domain and the Fc part most likely fold independently, both Fc domains could be used to obtain stable fusion proteins and neither the interaction of ACE2 with the virus spike protein nor its enzymatic function was affected. Thus, engineering general features of the fusion protein is possible without interfering with spike protein interaction.

The presence of the Fc domain could markedly increase the plasma half-life of ACE2-Fc due to the interaction of the Fc domain with the neonatal Fc-receptor (FcRn), and therefore a slower renal clearance of the fusion molecule. FcRn is broadly expressed on many cell types including endothelial cells and respiratory epithelial cells (Latvala et al., 2017; Sockolosky and Szoka, 2015) .

Binding to FcRn could extend the systemic half-life by chaperoning bound Fc fusion proteins away from lysosomal degradation. In addition, FcRn transports IgG and Fc fusion molecules across mucosal barriers into the lumen of the respiratory and intestinal tract thereby providing dynamic trafficking between circulating and luminal IgG molecules at mucosal sites (Sockolosky and Szoka, 2015; Tzaban et al., 2009 ). Here we showed that the ACE2-IgG4-Fc has preserved binding to FcRn with the same affinity as the ACE2-IgG1-Fc counterparts. On the other hand, the ACE2-IgG4-Fc did not bind to CD16 (FcRIIIa) in contrast to ACE2-IgG1-Fc, although the constant heavy chain regions of the different IgG subclasses share over 95% sequence homology. IgG4 in particular has poor ability to engage C1q and Fc gamma receptors and has been associated with anti-inflammatory properties. Most importantly, using our design, ACE2-IgG4-Fc fusion constructs J o u r n a l P r e -p r o o f show equally high binding affinity to the SARS-CoV-2 RBD and spike neutralizing the virus with IC50 values in the picomolar range.

The SARS-CoV-2 pandemic has caused an unprecedented challenge to develop COVID-19

therapies. However, progress with antiviral drugs has been slow. Here we show that ACE2-IgG4-Fc fusion proteins have favorable biophysical and pharmaceutical characteristics and significant in vitro SARS-CoV-2 neutralizing potency. The Fc part from IgG4 could bring clinical benefits for ACE2-Fc proteins by avoiding potential antibody-dependent disease enhancement during treatment. In addition, our data showed that ACE2-IgG4-Fc fusion proteins displayed increasing neutralizing potential, the further the SARS-CoV variant adapted to its human host becoming more infectious or escaping immune responses. For the currently emerging SARS-CoV-2 VoCs, alternative therapies are urgently needed, and ACE2-Fc fusion proteins certainly are interesting candidates. Thus, our ACE2-IgG4-Fc fusion protein is a promising candidate not only for therapeutic use in the current SARS-CoV-2 pandemic including more infectious SARS-CoV-2 VoC but also for future coronavirus infectious diseases. Upcoming studies will reveal whether the promising results presented herein can be translated to a therapeutic efficiency in vivo.

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Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Human recombinant soluble ACE2 in severe COVID-19

The authors like to thank Polpharma Biologics Utrecht B.V. for performing the transient transfections and providing the fusion molecules. SARS-CoV-2 Spike S1 Inhibition ELISA was performed in collaboration with TebuBio, France. Binding to Fc receptors was performed in collaboration with Vela Laboratories, Austria. We are grateful to Volker Thiel, University of Bern, 

J o u r n a l P r e -p r o o f

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:A patent application has been filed by Formycon AG for the content disclosed in this study. The authors A.R., F.-P. W., N.S., S.P., F.W., and C.B. are employees of Formycon AG. J.B. is advisory board member of Formycon AG. U.P. is shareholder and member of the board of SCG Cell Therapy seeking a license from Formycon to co-develop the product. Remaining authors declare no conflict of interest.