key: cord-0741353-geor69b4 authors: Castilho, Alexandra; Schwestka, Jennifer; Kienzl, Nikolaus F.; Vavra, Ulrike; Grünwald‐Gruber, Clemens; Izadi, Shiva; Hiremath, Chaitra; Niederhöfer, Janine; Laurent, Elisabeth; Monteil, Vanessa; Mirazimi, Ali; Wirnsberger, Gerald; Stadlmann, Johannes; Stöger, Eva; Mach, Lukas; Strasser, Richard title: Generation of enzymatically competent SARS‐CoV‐2 decoy receptor ACE2‐Fc in glycoengineered Nicotiana benthamiana date: 2021-02-12 journal: Biotechnol J DOI: 10.1002/biot.202000566 sha: 4a88cc6c6ee12682c8526078897afb3463508152 doc_id: 741353 cord_uid: geor69b4 Human angiotensin‐converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS‐CoV‐2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2‐fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2‐Fc variant is glycosylated with mainly complex human‐type N‐glycans and functional with regard to enzyme activity, affinity to the SARS‐CoV‐2 receptor‐binding domain, and wild‐type virus neutralization. Angiotensin-converting enzyme 2 (ACE2), a metallopeptidase of the renin-angiotensin system involved in maintaining blood pressure homeostasis, is the primary host cell receptor for SARS-CoV-2 binding and cell entry. [1] Administration of high concentrations of recombinant soluble human ACE2 reduces virus entry into target host cells by competition with cellular ACE2 for binding to the SARS-CoV-2 spike protein. [2] A drawback of soluble ACE2 is the short plasma half-life that can, however, be overcome by fusion to the fragment crystallizable (Fc) of human immunoglobulin G. [3] A recombinant ACE2-Fc fusion protein efficiently neutralized pseudovirions pseudotyped with SARS-CoV-2 spike protein. [4] Since ACE2 is also the cell entry receptor for SARS-CoV-1, recombinant ACE2-Fc and engineered high-affinity variants thereof do not only have therapeutic potential to prevent SARS-CoV-2 infections but are also candidates for preventive treatments of future coronavirus outbreaks. [5] For the ongoing pandemic as well as for emerging viruses, it is highly important to establish flexible production systems for recombinant protein therapeutics such as ACE2-Fc that are required worldwide in high demands. In addition to treatment and prophylaxis, different ACE2 variants are essential components of diagnostic assays and test kits, for example, to characterize neutralizing antibodies or to screen for novel inhibitors that interfere with the binding of SARS-CoV-2 to ACE2. Since several years, plants are increasingly used as platforms to produce diagnostic reagents and pharmaceutical proteins. In this report, we demonstrate the production of a functional recombinant ACE2-Fc in glycoengineered plants. A DNA fragment coding for amino acids 18-615 of human ACE2 and codon optimized for expression in Nicotiana benthamiana (Thermo Fisher Scientific) was cloned into the BsaI sites of the magnICON vector pICHα26211 carrying the coding sequence for the barley α-amylase signal peptide and the Fc-domain of human IgG1. [6] 2.2 ACE2-Fc expression, purification, and characterization ACE2-Fc was produced by agrobacterium-mediated transient expression in leaves of N. benthamiana ΔXT/FT. Four days after infiltration, a total soluble protein extract was prepared and clarified as described previously. [7, 8] ACE2-Fc was purified by affinity chromatography using a 5 mL HiTrap Protein A column (Cytiva) and 0.1 M glycine-HCl (pH 3.5) for elution. Eluate fractions were immediately neutralized using 2 M Tris (pH 12.0), pooled, concentrated (Amicon Ultra-0.5 Centrifugal Filter Units, Millipore) and dialyzed against PBS (pH 7.4) at 4 • C overnight using SnakeSkin Dialysis Tubing (Thermo Fisher Scientific). Affinity-purified ACE2-Fc was subjected to size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column (Cytiva) equilibrated with PBS (pH 7.4) for polishing. SECmulti-angle light scattering (SEC-MALS) analysis was carried out as described recently. [9] Production of ACE2-Fc in HEK293 cells was performed as described previously. [5] ACE2-Fc separated by SDS-PAGE was detected by Coomassie Blue staining or immunoblotting with anti-human IgG (H+L)-horseradish peroxidase antibody (Promega). For deglycosylation, samples were incubated with Endoglycosidase H (Endo H) or peptide-N-glycosidase F (PNGase F) (New England Biolabs) according to the manufacturer's instructions. Binding assays was performed in triplicates. Data were evaluated using the Octet data analysis software version 11.1.1.39. ELISA assays were carried out as described. [10] Hydrolysis of (7-methoxycoumarin-4-yl)acetyl-Ala-Pro-Lys ( SARS-CoV-2 neutralization by ACE2-Fc was assayed by qRT-PCR analysis of virus-exposed Vero E6 cells as described. [2] The wild-type SARS-CoV-2 strain used was isolated from an infected Swedish individual (GenBank accession number MT093571). SEC-purified ACE2-Fc was sequentially digested with chymotrypsin and trypsin and the glycopeptides were subjected to LC-ESI-MS analysis as described. [8] 3 RESULTS Recombinant ACE2-Fc was produced in leaves from glycoengineered ΔXT/FT N. benthamiana using a viral expression vector ( Figure 1A ). [7] In total soluble protein extracts, the main band of ≈110 kDa corresponds to full-length glycosylated ACE2-Fc ( Figure 1B) . A faint band of ≈35 kDa was also detected, which indicates limited proteolytic release of the Fc domain. ACE2-Fc was purified from total soluble protein extracts by protein A affinity chromatography and then subjected to preparative SEC. In addition to the slowly eluting peak containing cleaved Fc (about 5% of the affinity-purified material), we detected three peaks that likely correspond to intact monomeric (10%), dimeric (80%), and oligomeric (5%) ACE2-Fc ( Figure 1C ). The dimer fraction was isolated and subjected to SEC analysis combined with MALS detection to determine its native molecular mass. The experi- (Table S1, Supporting Information). The reduced mobility of HEK293 ACE2-Fc ( Figure 1E ) is likely derived from differences in N-glycan processing. [11] After protein A purification the ACE2-Fc yield was ≈80 μg g −1 leaf fresh weight when extracted 4 days after infiltration, with 50 μg g −1 recovered as dimer after SEC purification. equally effective as HEK293 ACE2-Fc ( Figure 2D ). Together these data show that the recombinant ACE2 peptidase domain from ACE2-Fc is correctly folded and fully functional when produced in plants. Human ACE2 is heavily glycosylated and N-glycans play a role for the interaction with the RBD of SARS-CoV-2 spike protein. [11] The expressed ACE2 peptidase domain harbors six N-glycosylation sites ( Figure 1A ) and all of them are glycosylated in mammalian cells. [11] The reduced mobility on SDS-PAGE and the determined native molecular mass suggests that the plant-produced variant is also heavily glycosy- To characterize ACE2-Fc N-glycosylation in a site-specific manner, we digested purified dimeric ACE2-Fc and subjected the glycopeptides to LC-ESI-MS analysis. N-glycans could be detected on all sites, with the N-glycan profiles of all six glycopeptides being remarkably similar. The major peak corresponded to the complex N-glycan GlcNAc 2 Man 3 GlcNAc 2 (GnGn) ( Figure 3B ). In addition, smaller peaks due to oligomannosidic N-glycans (Man7-Man 7 GlcNAc 2 , Man8-Man 8 GlcNAc 2 , and Man9-Man 9 GlcNAc 2 ) were also present. While five sites were completely glycosylated, N90 displayed only partial occupancy (92 ± 3.5%, mean ± SD, n = 3) ( Figure 3C ). By contrast, HEK293-derived ACE2-Fc analyzed by the same glycoproteomic approach is fully glycosylated on all six N-glycosylation sites. As described by a previous report for soluble ACE2, the N-glycans from HEK293-produced ACE2-Fc are more extensively processed and display increased microheterogeneity compared to plant-produced ACE2-Fc ( Figure S1 , Supporting Information). [11] Engineered ACE2 lacking the N-glycan at N90 has a two-fold higher affinity to RBD. [5] It is therefore plausible that the higher affinity of ACE2-Fc from N. benthamiana for RBD results to some extent from the partial underglycosylation at this position. Alternatively, the less processed complex Nglycans lacking galactose and sialic acid may be favorable for RBD binding. These possibilities will be addressed in future studies. Here, we show that functional recombinant ACE2-Fc with high binding affinity for SARS-CoV-2 RBD can be efficiently produced in plants and may be used as receptor decoy to treat coronavirus infections in humans. Besides supporting fast and efficient purification from soluble protein extracts, the fusion of the Fc domain to the ACE2 peptidase domain is supposed to extend the in vivo half-life in therapeutic applications through binding to the neonatal Fc receptor. [12] The Fc domain enables also interactions with other Fc receptors present on immune cells. These interactions trigger Fc-mediated effector functions including mechanisms for clearance of viruses and virus-infected cells. Fusion to the full-length Fc region of human IgG1 retains cysteine residues required for homodimer formation. From a previous study with EPO-Fc, we know, however, that considerable amounts of the Fc domain are cleaved in planta resulting in a mixture of full-length fusion protein and the cleaved Fc domain. [13] Moreover, we have observed that the hinge region is particularly susceptible to cleavage by endogenous plant proteases. [14] To minimize proteolytic degradation of ACE2-Fc, we have therefore replaced the processing-prone part of the hinge region with a protease-resistant linker. Although the CPPC sequence from the hinge region involved in dimerization is missing in our fusion protein, the vast majority of ACE2-Fc was still present as a dimer and bound to SARS-CoV-2 RBD at least as well as ACE2-Fc produced in HEK293 cells. Human recombinant soluble ACE2 has been used successfully to treat a severe COVID-19 patient. [15] Recombinant ACE2-Fc could be used in a similar manner. While certain Fc-receptor mediated effector functions could further enhance its anti-viral activities, there are concerns that the Fc portion of ACE2-Fc may also pose a risk due to Fcγ receptor-mediated functions like antibody-dependent enhancement. [16] Whether the presence of the human Fc domain in ACE2-Fc is beneficial or has adverse consequences in vivo cannot be predicted and has to be explored in further studies using FcγR humanized mice and testing in humans. [17] ACKNOWLEDGMENT The authors declare no conflict of interest. Gerald Wirnsberger and Janine Niederhöfer are employees of Apeiron Biologics AG. The data that support the findings of this study are available from the corresponding author upon reasonable request. Nikolaus F. Kienzl https://orcid.org/0000-0001-8057-3930 Elisabeth Laurent https://orcid.org/0000-0002-5234-5524 Lukas Mach https://orcid.org/0000-0001-9013-5408 Richard Strasser https://orcid.org/0000-0001-8764-6530 SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2 Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects Neutralization of SARS-CoV-2 spike pseudotyped virus by recombinant ACE2-Ig Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2 Expression of human butyrylcholinesterase with an engineered glycosylation profile resembling the plasma-derived orthologue Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure Exploring site-specific N-glycosylation of HEK293 and plant-produced human IgA isotypes Steric accessibility of the cleavage sites dictates the proteolytic vulnerability of the anti-HIV-1 antibodies 2F5, 2G12, and PG9 in plants SARS-CoV-2 seroconversion in humans: A detailed protocol for a serological assay, antigen production, and test setup Virus-receptor interactions of glycosylated SARS-CoV-2 spike and human ACE2 receptor Novel ACE2-Fc chimeric fusion provides long-lasting hypertension control and organ protection in mouse models of systemic renin angiotensin system activation Generation of biologically active multi-sialylated recombinant human EPOFc in plants The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: Down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms Human recombinant soluble ACE2 in severe COVID-19. The Lancet An ACE2 microbody containing a single immunoglobulin Fc domain is a potent inhibitor of SARS-CoV-2 The role of IgG Fc receptors in antibody-dependent enhancement Additional supporting information may be found online in the Supporting Information section at the end of the article.