key: cord-0872548-2ceemuit
authors: Schepens, Bert; van Schie, Loes; Nerinckx, Wim; Roose, Kenny; Van Breedam, Wander; Fijalkowska, Daria; Devos, Simon; Weyts, Wannes; De Cae, Sieglinde; Vanmarcke, Sandrine; Lonigro, Chiara; Eeckhaut, Hannah; Van Herpe, Dries; Borloo, Jimmy; Oliveira, Ana Filipa; Catani, Joao Paulo; Creytens, Sarah; De Vlieger, Dorien; Michielsen, Gitte; Zavala Marchan, Jackeline Cecilia; Moschonas, George D.; Rossey, Iebe; Sedeyn, Koen; Van Hecke, Annelies; Zhang, Xin; Langendries, Lana; Jacobs, Sofie; ter Horst, Sebastiaan; Seldeslachts, Laura; Liesenborghs, Laurens; Boudewijns, Robbert; Thibaut, Hendrik Jan; Dallmeier, Kai; Velde, Greetje Vande; Weynand, Birgit; Beer, Julius; Schnepf, Daniel; Ohnemus, Annette; Remory, Isabel; Foo, Caroline S.; Abdelnabi, Rana; Maes, Piet; Kaptein, Suzanne J. F.; Rocha-Pereira, Joana; Jochmans, Dirk; Delang, Leen; Peelman, Frank; Staeheli, Peter; Schwemmle, Martin; Devoogdt, Nick; Tersago, Dominique; Germani, Massimiliano; Heads, James; Henry, Alistair; Popplewell, Andrew; Ellis, Mark; Brady, Kevin; Turner, Alison; Dombrecht, Bruno; Stortelers, Catelijne; Neyts, Johan; Callewaert, Nico; Saelens, Xavier
title: Drug development of an affinity enhanced, broadly neutralizing heavy chain-only antibody that restricts SARS-CoV-2 in rodents
date: 2021-03-18
journal: bioRxiv
DOI: 10.1101/2021.03.08.433449
sha: eb467b7ceb0080806fe97c3115fdeda927b9b2c7
doc_id: 872548
cord_uid: 2ceemuit

We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and −2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and −2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a disease that has rapidly spread across the planet with devastating consequences 1 . SARS-CoV-2 infections can be asymptomatic and mostly present mild to moderately severe symptoms. However, in approximately 10% of patients, COVID-19 progresses to a more severe stage that is characterized by dyspnea and hypoxemia, which may progress further to acute respiratory distress often requiring longterm intensive care and causing death in a proportion of patients. The ongoing inflammation triggered by the innate recognition of the SARS-CoV-2 virus 2 contributes to severe disease progression.

Prophylactic vaccine candidates have been developed at an unprecedented speed and viral spikeencoding mRNA and adenovirus-vectored vaccines have already obtained emergency approval in many countries 3, 4 . COVID-19 vaccines will become the cornerstone of controlling the pandemic yet will likely leave a significant part of the population insufficiently protected. Immunity may be short-lived and vaccine efficacy may be lower in the elderly, the age group that is most at risk of developing severe COVID-19 5, 6 . Limited vaccine availability in many countries, vaccine hesitancy and viral evolution to escape human immunity 7 are other factors of which the impact is currently uncertain. Hence, passive antibody immunotherapy with broadly neutralizing molecules, to prevent or suppress viral replication in the lower airways, will likely find an important place in rescuing COVID-19 patients. Indeed, the early development of sufficient titers of neutralizing antibodies by the patient correlates with avoidance of progression to severe disease 8 , and early administration of recombinant neutralizing antibodies or those present in high-titer convalescent plasma can avert severe disease [9] [10] [11] . An advantage of antibodies and antibody Fc-based fusions compared with small molecule drugs is their long circulatory half-life imparted by the FcRn-mediated recycling into the bloodstream, which provides for long term control of virus replication even after a single administration 12 .

Developed at an extraordinary pace, Regeneron's REGN10933/REGN10987 pair (Casirivimab/Imdevimab) and Eli Lilly's LyCoV555 (Bamlanivimab) recently obtained FDA emergency use approval for the treatment of mild to moderate COVID-19 in non-hospitalized adults and pediatric patients who are at risk of developing severe disease 10, 13 . Since its first emergence in humans at the end of 2019, however, SARS-CoV-2 viruses have acquired mutations that indicate further adaptation to the human host. Some of these SARS-CoV-2 variants are of concern (VOCs) because they are associated with increased transmissibility or contribute to evasion of host immunity 14 . These circulating mutant viruses often have acquired mutations in the receptor-binding motif (RBM), the region of the RBD that interacts with hACE2 that is also the target of most of the reported neutralizing antibodies in immune plasma and monoclonal antibodies derived from convalescent patients. As a consequence, these SARS-CoV-2 variants display increased resistance to neutralization by many convalescent plasma-derived monoclonal antibodies, including two of the three that have received emergency use approval in the USA 15, 16 . This reinforces the need to develop neutralizing antibodies that bind to regions of the SARS-CoV-2 spike that are under lower human immune pressure and that poorly tolerate mutations without compromising virus fitness. Such broadly neutralizing antibodies would also constitute valuable tools for our pandemic preparedness for future outbreaks of viruses in this family.

We recently reported the discovery and structural characterization of a VHH (the variable domain of a heavy chain-only antibody, also known as a Nanobody), named SARS VHH-72 (here abbreviated as VHH72), with SARS-CoV-1 and -2 neutralizing capacity 17 . VHH72-Fc was the first SARS-CoV-2 neutralizing antibody that binds to the RBD region that partially overlaps with the epitope of the anti-SARS-CoV-1 antibody CR3022, which is however not ACE2-competing and non-neutralizing against SARS-CoV-2. VHH72 binds to an epitope in the receptor-binding domain (RBD) of the spike protein that is highly conserved in members of the Sarbecovirus subgenus of the Betacoronaviruses 18, 19 , prevents the interaction of the SARS-CoV-1 and -2 RBD with ACE2, and, by trapping the RBD in the "up"

conformation, presumably destabilizes the spike protein of SARS-CoV-1 and -2 17 . Prophylactic administration of a prototype VHH72-human IgG1 Fc domain fusion strongly restricted SARS-CoV-2 replication in the lungs of experimentally infected golden Syrian hamsters 20 . The VHH72 contact region is occluded in the closed spike conformation, which is the dominant one on the native virus 21 . Even in the '1-RBD-up' conformation that can bind the ACE2 receptor, the epitope is positioned such that human monoclonal antibodies cannot easily reach it. Possibly because of this, amidst hundreds of antibodies against other regions of the spike, very few human antibodies have been reported that bind to an epitope that substantially overlaps the VHH72 epitope 22 . Moreover, the VHH72 contact region on the RBD is comprised of residues that form crucial packing contacts between the protomers of the trimeric spike. SARS-CoV-2 viruses with mutations in this contact region remain extremely rare.

Consistently, as we also illustrate in the present paper, none of the recently emerging and rapidly spreading VOC RBD mutations are in the VHH72 epitope.

Antibodies that cross-neutralize SARS-CoV-1 and -2 and other viruses of the Sarbecovirus subgenus, are very rare. Therefore, we decided to build on the VHH72 discovery, to generate a potency-enhanced drug designed for safety and long circulatory half-life, with a target product profile suitable for both treatment and prophylaxis of infections by SARS-CoV-2 and related viruses. This resulted in the design of the fully optimized XVR011, which is presently entering clinical development. Whereas the initial purpose is systemic administration to patients at risk of aggravated disease, we also show that nasal delivery of VHH72-Fc fully protects in a mouse model against SARS-CoV-2 challenge infection.

Many laboratories now have the capability to rapidly identify and characterize camelid single-domain VHH binders, either from in vivo immunizations or from selections from in vitro displayed repertoires, and the platform has hence been widely deployed in the present SARS-CoV-2 crisis 17,23-31 . However, a binder, no matter how potent, is not necessarily a drug. In a broader sense, our report on the entire lead molecule development track from our VHH72 pre-lead molecule to a fully optimized VHH-Fc drug substance, for the first time provides a detailed description of the development track that is needed from such prototype anti-viral VHH into a VHH-hIgG1-Fc antiviral clinical lead drug candidate, which may guide other such efforts and contribute to our pandemic readiness in the future.

Prototype VHH72-Fc protects K18-hACE2 mice from SARS-CoV-2 induced disease. We previously demonstrated protection in the Syrian hamster SARS-CoV-2 infection model by our prototype VHH72-Fc (here referred to as WT-VHH/12GS-WT-Fc; for an overview of the purified, CHO-produced VHH72-Fc constructs described in this study we refer to Supplementary Table 1) 20 . To gain more confidence, we also explored its protective efficacy in a mouse model expressing against SARS-CoV-2 induced disease ( Fig. 1d-f ). Viral titers in the lungs were also significantly reduced in WT-VHH/12GS-WT-Fc-treated mice when compared with the controls (Fig. 1d-f ). These data and our earlier report 20 indicated that the pre-lead prototype molecule with VHH72 specificity in the context of the half-life prolonging and bivalency-imparting hIgG1 Fc fusion had the potential to robustly protect against SARS-CoV-2 virus challenge in a prophylactic setting in two different animal models following parenteral and intranasal administration.

VHH72 binds a highly conserved region in the RBD of SARS-CoV-2. VHH72 binds to a region in the core of the RBD that is distal from the much more variable RBM 17 . Free energy contribution analysis by FastContact 35 of snapshots from Molecular Dynamics simulations with the VHH72-RBD complex indicates that the epitope recognized by VHH72 has a prominent two-residue hot-spot, consisting of F377 and K378, which contact VHH72 residues V100 and D100g, respectively ( Supplementary Fig. 1a ).

The epitope is exposed only when the trimeric spike protein has at least one RBD in an 'up' conformation ( Supplementary Fig. 1b) 17 . In the three-RBD 'down' state, the VHH72 contact region belongs to an occluded zone that makes mutual contacts with the adjacent RBDs ( Supplementary Fig.   1c ), as well as with the helix-turn-helix positioned between heptad repeat 1 and the central helix of the underlying S2 domain. These subtle inter-RBD and inter-S1/S2 contacts allow oscillation between the RBD 'down' and the ACE2-engaging 'up' positioning 36, 37 . Presumably due to this involvement in spike conformational dynamics, the amino acid residues that contribute to the VHH72 contact region are remarkably conserved in circulating SARS-CoV-2 viruses ( Supplementary Fig. 2 ). Moreover, deep mutational scanning analysis has shown that the VHH72 contact region overlaps with a patch of the RBD in which mutations may severely compromise the fold 38 , further explaining why it is indeed a highly conserved region on the Sarbecoviral RBD.

Design and selection of a VHH72 variant with increased neutralizing activity. While the prototype VHH72-Fc molecule had promising in vivo efficacy, we wished to further enhance its potency, with an eye towards ease of administration at reduced dosage through various routes, including subcutaneous and inhaled. Given the limited available time in this emergency lead development campaign, we decided on a protein modeling based mutant design approach for a VHH72 variant with increased affinity for SARS-CoV-2 RBD. At the start of our investigation, immediately after the SARS-CoV-2 genome was published, however, no SARS-CoV-2 RBD structure was publicly available. Therefore, based on the crystal structure of VHH72 in complex with the SARS-CoV-1 RBD (PDB code: 6WAQ), a model of the SARS-CoV-2 RBD was generated through the I-TASSER server 39 , which was then superimposed by means of the Swiss-PdbViewer 40 to the SARS-CoV-1 RBD/VHH72 structure (Fig. 1g ).

Only three residues are different between SARS-CoV-2 and -1 at the VHH72-RBD interface: (1) A372

(T359 in SARS-CoV-1), resulting in the loss of a glycan on N370 (N357 in SARS-CoV-1); (2) N439 (R426 in SARS-CoV-1), resulting in the loss of an ionic interaction with VHH72 residue D61; and (3) P384 (A371 in SARS-CoV-1). Close to P384 is Y369, which is a key contact residue for VHH72 ( Supplementary Fig.   1 ), for which I-TASSER predicted an upward conformation in the SARS-CoV-2 RBD-VHH72 model (Fig.   1g ). In the SARS-CoV-1 RBD-VHH72 co-crystal structure, however, the corresponding Y356 points downward and resides in a groove-like depression between two short helices of the RBD. The upconformation of SARS-CoV-2 Y369 sets it in a mostly hydrophobic small cavity of VHH72, contacting residues S52, W52a, S53, S56 (all in CDR2) and V100 (in CDR3) (Fig. 1g) . Molecular dynamics simulations with Gromacs 41 shows that Y369 can be readily accommodated in that cavity. Interestingly, the later reported cryo-EM or crystal structures of SARS-CoV-2 RBD indeed typically show Y369 in the upward conformation (e.g. PDB-entries 6VSB, 6M17, and 6VXX) [42] [43] [44] . Presumably Y369, as well as its Y356

counterpart in SARS-CoV-1 RBD, can flip into up or down positions, with the up-position prevailing in SARS-CoV-2 RBD due to a conformational constraint imposed by the nearby P384, which is A371 in SARS-CoV-1.

Guided by molecular modeling, we generated a set of VHH72 variants with point mutations in the residues that line the cavity that accommodates Y369 of SARS-CoV-2 RBD. To ensure that we would only prioritize mutations that enhanced affinity in our intended bivalent Fc-fusion drug context, these variants were rapidly prototyped in parallel as Fc fusions in Pichia pastoris, which can produce VHH-Fc fusions much more easily than native hIgG1s. The crude yeast medium was screened for binding to immobilized SARS-CoV-2 RBD by biolayer interferometry (BLI). Introduction of S56A resulted in a slower off-rate in this bivalent context ( Supplementary Fig. 3a ,b). VHH_S56A/12GS-WT-Fc also displayed higher binding affinity for SARS-CoV-2 spike expressed on the surface of 293T cells than parental WT-VHH/12GS-WT-Fc ( Supplementary Fig. 3c ).

Next, we humanized the monovalent VHH72 with or without the S56A mutation, based on a sequence comparison with the human IGHV3-JH consensus sequence, and the N-terminal glutamine was replaced by a glutamic acid codon to avoid N-terminal pyroglutamate formation, in order to increase the chemical homogeneity ( Supplementary Fig. 4 ). As with its Fc fusion, we validated that the S56A substitution increased the affinity of the monomeric humanized VHH72 for immobilized SARS-CoV-2 RBD, by approximately seven-fold as determined by BLI (Fig. 1h ). Increased affinity of humVHH_S56A for prefusion stabilized SARS-CoV-2 spike was also observed in ELISA and for SARS-CoV-2 spike expressed on the surface of mammalian cells by flow cytometry (Fig. 1i,j) . In addition, the mutant prevented the binding of SARS-CoV-2 RBD to ACE2 on the surface of VeroE6 cells seven times more efficiently than humVHH (Fig. 1k ). This improved affinity correlated with significantly stronger neutralizing activity of Hum_S56A as determined in a VSV SARS-CoV-2 spike pseudotyped virus neutralization assay (IC50 humVHH: 2.550 µg/ml; IC50 humVHH_S56A: 0.837 µg/ml) (Fig. 1l) .

Importantly, humVHH_S56A also neutralized VSV SARS-CoV-1 spike pseudotypes with 10-fold higher potency and bound with higher affinity to SARS-CoV-1 RBD and spike than humVHH (IC50 humVHH: 0.491 µg/ml; IC50 humVHH_S56A: 0.045 µg/ml) ( Fig. 1m and Supplementary Fig. 5 ).

VHH72_S56A-Fc constructs with potent SARS-CoV-2 neutralizing activity. We opted to include a human IgG1 with minimal Fc effector functions in our VHH72-Fc designs because there is uncertainty about the possible contribution of IgG effector functions to disease severity in COVID-19 patients [45] [46] [47] .

To this effect, and as also chosen by several other anti-SARS-CoV-2 antibody developers 48, 49 , we opted to use the well-characterized LALA mutations in the Fc portion, with or without the P329G mutation (LALAPG) [50] [51] [52] . In an initial set of experiments, we validated that neither the Gly-Ser linker length between the VHH and the Fc hinge (2 or 14 amino acids), nor the humanization of the VHH nor the introduction of LALAPG mutations in the Fc affected the affinity for SARS-CoV-2 S or its RBD, as determined by BLI, ELISA, flow cytometry and an ACE2 competition assay (Fig. 2a -e and Supplementary   Fig. 6b ,c). Consistent with this, potency (PRNT50) in a plaque neutralization assay using authentic SARS-CoV-2 virus was unaffected by these changes in the linker length or the Fc part (Supplementary Table   1 ). Subsequently, we built the S56A humanized VHH variants of the WT and LALAPG-Fc-fusions.

Consistent with the results obtained with the Pichia-produced prototypes, we observed 2-3-fold higher affinity for immobilized bivalent SARS-CoV-2 RBD as determined in BLI, which was further confirmed in flow cytometry-based quantification assays using mammalian cells that express spike on their surface, in hACE2-Fc RBD competition AlphaLISA, and in a VeroE6 cell-based SARS-CoV-2 RBD competition assay (Fig. 2a- ileum (2 log) was observed on day 4 after infection in both VHH72-Fc treated groups, and in stool samples also for humVHH_S56A/LALAPG-Fc (Fig. 2h) . Protection was also evident based on µ-computer tomography (µCT) imaging of the lungs on day 4, which showed a significantly reduced incidence of dilated bronchi in the VHH72-Fc treated animals (Fig. 2i) . As both methods of affinity enhancement worked well in this experiment, both the bivalent and tetravalent designs were propagated in the subsequent stage of the drug development campaign.

Generation of a lead therapeutic. Robust expression levels, chemical and physical stability, and absence of atypical posttranslational modifications are important prerequisites for the "developability" of a biologic 55 . Finalizing the design of our molecules for optimal homogeneity, we first truncated the upper hinge of the Fc, in common with most Fc fusions, which avoids heterogeneity due to an unpaired cysteine residue, and compensated for the reduced spacing between the VHH and the Fc by extending the glycine-serine linker to 10 amino acids: (G4S)2. Next, analysis by mass spectrometry had revealed partial C-terminal lysine removal in CHO-produced product, and we Supplementary Fig. 11b ). The tetravalent molecule also had a more acidic pI that would be potentially less favorable for a manufacturing strategy, requiring greater optimization of ion exchange steps (Supplementary Table 7 (Fig. 4a,b) . A clear dose-response relationship was observed, with increased variability in responses at the 2 mg/kg doses (Fig. 4a,b) . Gross lung pathology was lowest in the animals that had been treated with 7 mg/kg of the bivalent construct (Fig. 4c ). Viral loads in the bronchoalveolar lavage fluid of the hamsters were reduced to background levels in all but 2 animals that had received humVHH_S56A/LALAPG-Fc/Gen2 or (humVHH_S56A)2/LALAPG-Fc/Gen2

( Supplementary Fig. 12a ). Interestingly, viral titers in the nose and throat of the challenged hamsters were also significantly and dose-dependently reduced compared with the palivizumab control group, indicating that parenteral, post-challenge administration of the VHH-Fc constructs restricts viral replication in the upper and lower respiratory tract of the hamsters ( Supplementary Fig. 12b,c) .

In a separate experiment, we independently validated this therapeutic dose finding for the bivalent and tetravalent VHH-Fc fusion lead constructs at 7 and 1 mg/kg at a different laboratory (Neyts lab) by intraperitoneal injection, 16h after challenge with a different SARS-CoV-2 isolate (BetaCov/Belgium/GHB-03021/2020 strain) (Fig. 4d) . As a prophylactic control, the bivalent humVHH_S56A/LALAPG-Fc/Gen2 construct was administered 1 day prior to challenge at 7 mg/kg. In this study, the infectious virus load in the lungs was also here significantly reduced compared with the control treated animals for both the humVHH_S56A/LALAPG-Fc/Gen2 and (humVHH_S56A)2/LALAPG-Fc/Gen2 treated groups at 7 mg/kg, but not at the 1 mg/kg treatment with the bivalent construct. In At this point, we considered whether to choose the bivalent or tetravalent molecule for further development. While in vitro, the tetravalent molecule is clearly more potent, this difference is not apparent in vivo. Tetravalency moreover came at a cost of a more complex molecule to manufacture and stably formulate, as described above. We hence prioritized the simpler bivalent design and selected humVHH_S56A/LALA-Fc/Gen2 as our clinical lead molecule, which was termed XVR011.

humans upon systemic administration, antibodies have to have a low propensity for off-target binding to other human membrane/extracellular proteins. To evaluate this for XVR011, we used a human membrane protein microarray assay in which reactivity was probed against fixed HEK293 cells that each overexpress one of 5475 full-length human plasma membrane proteins and cell surface-tethered human secreted proteins and a further 371 human heterodimeric such proteins 58 . Only four proteins were found to potentially show some binding in a high-sensitivity primary screen using XVR011 as primary antibody and an anti-human IgG1 secondary detection antibody: over-expressed proteins FCGR1A, IGHG3, IGF1, and CALHM6, next to the spotted SARS-CoV2 Spike protein, the primary target and positive control. In a targeted confirmation experiment to study binding to these proteins in more detail, we used the clinically well-validated rituximab (anti-CD20) and cells expressing its antigen as a control for potential hIgG1 Fc-mediated interactions, as well as a buffer control instead of primary antibody, to check for interactions with the secondary detection antibody ( Supplementary Fig. 13 ).

Apart from the expected XVR011 reactivity with recombinant SARS-CoV-2 spike protein, binding to the primary hits was as low or lower as that of rituximab (which bound CD20-expressing cells) and a robust detection signal was only observed for fixed cells that expressed human immunoglobulin heavy gamma-3 chain IGHG3. However, this reactivity was equally strong with the PBS and rituximab controls, and, therefore due to direct binding of the secondary detection antibody. Based on these results, we conclude that XVR011 is very specific and not polyreactive to human proteins at all, which supports its potential for safe use as a medicine.

Further validating the built-in safety feature, we verified that the introduced LALA mutations in the context of VHH-Fc fusion construct XVR011 resulted in reduced binding to activating Fc receptors. We 

There is an urgent need for safe and effective anti-SARS-CoV-2 drugs that can prevent or cure COVID-19. We report the protein engineering-based drug development of a potent cross-neutralizing, VoC- ACE2 binding. The much lower immunogenicity of the VHH72 binding region in humans than the epitopes on the RBM is also consistent with a recent large-scale serological survey of convalescent SARS-CoV-2 patients, which demonstrated that mAbs with epitopes strongly overlapping that of VHH72 (site II in that study) were competed against much less potently and were present in a smaller proportion of patient sera than was the case for mAbs targeting the ACE2-binding region of the RBD 22 .

These results were very similar to what was observed for S309, which is also a SARS-CoV-1/2 crossneutralizing antibody presently in clinical development 59 . Amongst these binding agents presently described against this 'cryptic supersite', XVR011 best combines a very strong potency both in vitro and in vivo, SARS-CoV-1/2 cross-neutralization, very broad cross-clade sarbecovirus binding, and unaltered strong potency against the currently emerging VoCs, warranting its clinical development.

For this, we considered it prudent in patients with progressing COVID-19 disease to mainly rely on a pure virus neutralization mechanism of action, and thus to suppress Fc receptor binding of the antibody's Fc domain. Interestingly, antibodies that bind to VHH72-epitope overlapping epitopes, by nature already have very low complement-dependent cytotoxicity because this RBD region is not sterically accessible in the dominant closed spike conformation of the native spike protein 22 . To silence antibody Fc-mediated effector functions, we settled on the IgG LALA-Fc mutations, which are amongst the best-validated for this purpose 52 . While the field of SARS-CoV-2 antibody development currently sees arguments in both directions with regard to the desired extent of Fc effector functionality in diverse settings (prophylactic, therapeutic, route and frequency of administration), our LALA-Fc choice attempts to strike an optimal balance between safety and efficacy in diverse application modalities, within the limitations of relevant current clinical data, while ascertaining an established regulatory track record and freedom to operate.

It was recently reported that in a therapeutic setting, some human neutralizing antibodies require intact Fc effector functions to control SARS-CoV-2 replication in the K18-hACE2 transgenic mouse and Syrian hamster challenge models 68 , using the LALAPG mutations to remove effector function of those antibodies. In contrast to that report, we found that Fc effector silent humVHH_S56A/LALA(PG)- for therapeutic efficacy appears to be very antibody-dependent even with human antibodies, as a very recent study also demonstrated therapeutic efficacy in the same hamster model of other RBM-binding LALAPG-modified antibodies 69 .

The pharmaceutically fully developed VHH72-based biologic named XVR011, that combines potent neutralizing activity with high stability, broad coverage and silenced Fc effector functionality for enhanced safety, has currently completed cGMP-manufacturing and formal preclinical development.

Clinical studies are now being started to evaluate safety and efficacy of rapid administration upon hospitalization of patients, within the first week of COVID-19 symptoms.

We Expression of the surface-displayed myc-tagged RBDs was detected using a FITC conjugated chicken anti-myc antibody (Immunology Consultants Laboratory, Inc.). Following 3 washes with PBS containing 0.5% BSA, the cells were analyzed by flow cytometry using an BD LSRII flow cytometer (BD Biosciences).

The binding curves were fitted using nonlinear regression (Graphpad 8.0). 

The main data supporting the findings of this study are available in the paper and its Supplementary information. The associated raw data are too numerous to be readily shared publicly and can be made available from the corresponding authors upon reasonable request. Table 6 ). The protein conjugate analysis was performed based on the differential extinction coefficients and refractive index values of proteins versus conjugated glycan modifiers. Binding affinity of VHH72-Fc variants to immobilized mouse Fc-fused SARS-CoV-2 RBD (RBD-mFc). Apparent kinetics of the 2:2 interaction is based on a global 1:1 fit of the replicate (n = 2) data; values are the averages of replicates. b. Binding of the indicated VHH72-Fc constructs (see Supplementary Table 2 for a description) to coated SARS-CoV-2 spike determined by ELISA (data points are mean ± SD; n=3). c. Binding of the indicated VHH72-Fc constructs to cell surface expressed SARS-CoV-2 spike determined by flow cytometry. The graph shows the mean (n=2) ratio of the MFI of transfected (GFP + ) cells over the MFI of non-transfected (GFP -) cells. d. Inhibition of ACE-2/RBD interaction determined by AlphaLISA (amplified luminescent proximity homogeneous assay). Biotinylated SARS-CoV-2 RBD was loaded on streptavidin coated Alpha Donor beads and human ACE-2-mFc protein was captured on anti-mouse IgG acceptor beads. Interference of the donor-acceptor bead interaction was assessed for serial dilutions of the indicated VHH-Fc constructs. Graph pad Prism was used for curve fitting and IC 50 

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A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2

In vitro characterization of five humanized OKT3 effector function variant antibodies

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility

First cases of coronavirus disease 2019 (COVID-19) in the WHO European Region

Biophysical properties of the clinical-stage antibody landscape

Developability assessment during the selection of novel therapeutic antibodies

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

New Advances in Cell Microarray Technology to Expand Applications in

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

Early transmissibility assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom

SARS-CoV-2 RBD in vitro evolution follows contagious mutation spread, yet generates an able infection inhibitor

Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity

Emergence and rapid spread of a new severe acute respiratory syndromerelated coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa

SARS-CoV-2 reinfection by the new Variant of Concern (VOC) P.1 in Amazonas, Brazil -SARS

CoV-2 coronavirus / nCoV-2019 Genomic Epidemiology

Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody

Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections

Structural basis of a shared antibody response to SARS-CoV-2

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection

Extremely potent human monoclonal antibodies from COVID-19 convalescent patients

Improved side-chain torsion potentials for the Amber ff99SB protein force field

Combinatorial optimization of

CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris

Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris

A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly

High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol

On the distribution of protein refractive index increments

Characterization of group C meningococcal polysaccharide by light-scattering spectroscopy. III. Determination of molecular weight, radius of gyration, and translational diffusional coefficient

A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Comparative infectivity and pathogenesis of emerging SARS-CoV-2 variants in Syrian hamsters

Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Naunyn-Schmiedebergs Arch

High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method

Favipiravir at high doses has potent antiviral activity in SARS-CoV-2-infected hamsters, whereas hydroxychloroquine lacks activity