Converting non-neutralizing SARS-CoV-2 antibodies into broad-spectrum inhibitors

Omicron and its subvariants have rendered most authorized monoclonal antibody-based treatments for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ineffective, highlighting the need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective antibodies target variable epitopes. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to known non-neutralizing antibodies that target highly conserved epitopes in the viral spike protein. These inhibitors, called receptor-blocking conserved non-neutralizing antibodies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOCs), including Omicron. Neutralization potency is lost when the linker joining the binding and inhibitory ReconnAb components is severed. In addition, a bi-functional ReconnAb, made by linking ACE2 to a bi-specific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron and BA.2. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad-spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

T he emergence of the Omicron variant has rendered six of the seven 1,2 clinically available monoclonal antibodies (mAbs) essentially ineffective against SARS-CoV-2; only sotrovimab retains robust neutralizing activity against Omicron 2,3 . These clinical mAbs all target the receptor binding-domain (RBD) 1 of the spike protein and were selected for their neutralizing potency against Wuhan-Hu-1 SARS-CoV-2. The six mAbs besides sotrovimab target non-conserved (variable) regions of the RBD 4-7 and prevent interaction with its receptor, ACE2 (refs. 6,8,9 ). Sotrovimab, a derivative of the mAb S309 (ref. 10 ), was initially isolated from a survivor of SARS-CoV-1, so its epitope in the RBD is more highly conserved 11 , although in vitro escape mutations have been identified 5 . Moreover, since this article has been in review, sotrovimab has lost significant activity against the recent BA.2 variant 12 . This has necessitated authorization of a new mAb, bebtelovimab, which is capable of neutralizing BA.2 (ref. 13 ).
The spike protein is large (~450 kDa as a trimer) and contains extensive regions that are extremely highly conserved (Fig. 1a). Some residues on the spike that are distant from the RBD have near-perfect sequence identity within related coronaviruses (Fig. 1b). Presumably, these regions are highly conserved because they are required for viral activity (for example, membrane fusion) 14 . Although the spike protein of Omicron has a much larger mutational profile than that of previous VOCs 15 -with 36 total mutations, 15 being in the RBD 2,16 -the highly conserved epitopes remain largely unaltered ( Fig. 1) 2,16 .
In other viral spike proteins-for instance, influenza hemagglutinin 17-19 -highly conserved epitopes outside of the receptor-binding region are targets of potent broadly neutralizing antibodies (bnAbs). However, despite the heightened interest sparked by the global pandemic, the search for bnAbs against betacoronaviruses has been largely disappointing. Although one conserved helical epitope at the base of the spike protein has been shown to elicit rare mAbs with relatively broad neutralizing activity, their potency is often weaker than RBD-directed neutralizing Abs [20][21][22] . Furthermore, neutralizing N-terminal domain (NTD) 23 antibodies have been identified, but their epitopes are not highly conserved.
Indeed, available evidence suggests that conserved regions outside the RBD generally elicit non-neutralizing mAbs 21,[24][25][26][27][28][29] . We hypothesized that we could generate potent, broad-spectrum inhibitors by modifying existing non-neutralizing antibodies, which target highly conserved epitopes on the spike protein, to also contain a receptor-blocking component. Due to the conservation of their epitopes, such inhibitors would potentially be broadly neutralizing.
Here we introduce ReconnAbs (pronounced 'recon-abs'), a novel class of therapeutic proteins in which non-neutralizing antibodies that target highly conserved, non-RBD epitopes are tethered to the ACE2 receptor, which otherwise has low intrinsic affinity and neutralizing potency. The cross-reactive, non-neutralizing antibodies were identified in a two-step process. First, we analyzed the phylogenetic trees of a collection of SARS-CoV-2 antibodies and eliminated those that are likely to bind the RBD. Then, similarly to the development of sotrovimab 11 , we determined which of these non-RBD antibodies bound to the SARS-CoV-1 spike protein. We predict that ReconnAbs will have increased potency due to the increase in effective concentration of each component 30 , as has been shown previously for bi-specific antibody fusions 31 and antibody-ACE2 fusions 32 . More importantly, ReconnAbs are predicted to have increased broad-spectrum activity by targeting highly conserved, non-RBD epitopes on spike. We demonstrate that ReconnAbs show neutralizing activity against all SARS-CoV-2 VOCs tested, including Omicron and BA.2. Furthermore, a bi-specific ReconnAb containing two non-neutralizing antibodies with non-overlapping epitopes fused to ACE2 confers sub-nanomolar neutralization against all throughout the trees ( Fig. 2a and Extended Data Fig. 1). These included at least one antibody sequence from all clusters containing four or more non-RBD-binding antibodies. The sequences we chose based on their HC sequences also showed diversity in the LC phylogenetic tree (Extended Data Fig. 1). These 48 clones display a range of CDRH3 and CDRL3 lengths (Fig. 2b) and use an array of V-genes in both the HC and LC (Fig. 2c), further confirming their diversity.
To determine which of these 48 non-RBD-binding antibodies target highly conserved epitopes, we used binding to the SARS-CoV-1 spike as a surrogate for epitope conservation. We designed the 48 single-chain variable fragment (scFvs) constructs by fusing the antibody HC and LC variable regions to the yeast surface protein Aga2p to enable yeast surface display. To profile the scFv panel, we optimized production of biotinylated SARS-CoV-2 and other human coronavirus (hCoV) spike proteins (Extended Data Fig. 2a-d) and produced biotinylated versions of the SARS-CoV-2 and SARS-CoV-1 spike proteins. These were used to probe the yeast library by fluorescence flow cytometry (Fig. 2d). The complete 48-member library showed robust (82%; Fig. 2d) staining with the SARS-CoV-2 spike, consistent with the original antibody collection having been isolated from SARS-CoV-2 convalescent donors. The library had reduced (21%; Fig. 2d) staining with the SARS-CoV-1 spike proteins overlaid on the SARS-CoV-2 spike protein structure (left) and the SARS-CoV-2 RBD (right; residues 319-541) (PDB ID: 6VXX) identifies a highly conserved patch in S2. Color gradient is a step gradient of conservation containing nine total steps of sequence conservation identified from the ConSurf database; gradient is shown on the bottom. b, Sequence identity for all residues in the SARS-CoV-2 spike protein compared to a set of 44 related coronavirus spike proteins shows higher conservation in the S2 relative to the S1. A value of 1.0 means perfect identity across all compared coronavirus proteins. RBD and NTD domains of SARS-CoV-2 spike are labeled on the top; S1 and S2 domains are labeled on the bottom. spike. Consistent with the intention of the library, no clones bind to the RBD of SARS-CoV-2 (Fig. 2d).
Having identified 48 antibodies that bind outside the RBD, we next selected those that bind to highly conserved regions of the spike protein. To do this, we used fluorescence-activated cell sorting (FACS) 34 with the SARS-CoV-1 spike protein as bait (Extended Data Fig. 3) and identified ten sequences. We confirmed by ELISA that the corresponding full-length IgG antibodies (Extended Data Fig. 4a) bind to both SARS-CoV-2 and SARS-CoV-1 spike proteins (Extended Data Fig. 4b). Of the ten, seven clones were strong SARS-CoV-1 binders, confirmed by biolayer interferometry (BLI) (Fig. 2e). One clone, COV2-2449, also binds MERS and OC43 spike proteins (Extended Data Fig. 4b,c). No clones bind to the NTD (Extended Data Fig. 4c). Consistent with previous reports 33, [35][36][37][38] , these antibodies were non-neutralizing in our assay (Extended Data Fig. 5).
We used BLI to characterize the epitopes of these seven antibodies in a binding competition assay. We loaded each antibody onto either SARS-CoV-2 or SARS-CoV-1 spike proteins, and then we tested for subsequent binding of each of the other antibodies (Fig. 2f). The results suggest that there are five primary epitopes, of which four are in a partially overlapping supersite (Fig. 2f), likely corresponding to the extensive, continuous patch of highly conserved residues on the spike protein surface (Fig. 1a). Two sets of antibodies had indistinguishable epitopes: the pair 38 of COVA2-14 and COVA2-18 and the pair of CV27 and COV2-2147. This result is consistent with the phylogeny, which shows the antibodies in the two pairs clustered very closely together ( Fig. 2a and Extended Data Fig. 1). The identification of five unique epitopes from the seven selected antibodies highlights the diversity in the initial starting library.
scFv-based ReconnAb development. We selected five antibodies, one from each described epitope (Fig. 2f,g), and converted these non-neutralizing, cross-reactive antibodies into ReconnAbs by fusion to the ACE2 ectodomain, as the receptor-blocking component of the ReconnAb design. We designed the linker to be long enough to allow for simultaneous binding of both ACE2 to the RBD and the scFv, regardless of epitope, to the spike S2 domain ( Fig. 3a and Extended Data Fig. 6a). We joined the C-terminus of the scFv to the N-terminus of ACE2, because the N-terminal residue of the ACE2 ectodomain is adjacent to the SARS-CoV-2 RBD when bound. We also incorporated within the linker a hexa-histidine tag for purification and a TEV protease site to enable assessment of ReconnAb activity when its binding and inhibitory components are separated ( Fig. 3a and Extended Data Fig. 6a). We anticipated that ReconnAbs would bind to both a highly conserved site on the spike protein and, simultaneously, to the RBD through the ACE2 domain (Fig. 3b). However, if cleaved at the TEV site, the intrinsically low-affinity ACE2 domain would not benefit from the affinity of the non-neutralizing antibody (Fig. 3b).
We expressed and purified the five ReconnAbs and used gel electrophoresis to confirm that TEV cleavage separated the ACE2 and scFv components (Fig. 3c). BLI experiments showed that TEV cleavage of the ReconnAb proteins reduced binding to both SARS-CoV-2 and SARS-CoV-1 spike proteins (Fig. 3d), consistent with the lower affinity of monomeric ACE2 (ref. 39 ). We then investigated the ability of the ReconnAbs to block ACE2 binding to the SARS-CoV-2 spike protein. ACE2 competition is often used as a surrogate for neutralization, as preventing ACE2 binding prevents the virus from interacting with target cells. Indeed, uncleaved ReconnAbs show substantial interference with binding of an Fc version of human ACE2 (hFc-ACE2), whereas TEV-cleaved ReconnAbs do not (Fig. 3e).
We next investigated if ReconnAbs were able to neutralize lentiviral pseudoviruses corresponding to the SARS-CoV-2 VOCs and found that all ReconnAbs neutralized all VOCs, some showing nanomolar potency against Omicron (Fig. 4a). Consistent with its lower affinity (Fig. 3g), COV2-2143-ACE2 had the weakest neutralization of the tested ReconnAbs (Fig. 4a). COV2-2449-ACE2 showed the least deviation in neutralization potency among variants, consistent with its epitope being the most highly conserved (Extended Data Fig. 4b,c). Moreover, in live virus assays, CV10, CV27 and COV2-2449 showed neutralization against Wuhan-1 and Omicron SARS-CoV-2 virus (Supplementary Table 1). Neutralization is, as expected, slightly lower in this format given the use of a limiting dilution assay 40 . Notably, the TEV-proteolyzed versions of the ReconnAbs did not confer the same neutralizing potency as their uncleaved counterparts (Fig. 4a), demonstrating that the separate components are not working synergistically, but that the tether is essential for the ReconnAb components to work cooperatively. To examine if our selected linker length was sufficient to confer the desired activity, we investigated an additional longer linker length for two scFv-ACE2 fusions, from two distinct epitopes. Our CV27-ACE2 and COVA2-14-ACE2 fusions did not show any improved neutralization with an additional seven amino acids in the linker, suggesting that the examined linker length is sufficient to confer the desired activity (Extended Data Fig. 6b-d).

Bi-functional, IgG-based ReconnAb development. Two
ReconnAbs, CV10-ACE2 and COV2-2449-ACE2, were of particular interest as they showed broad-spectrum neutralization (Fig. 4a) and did not have overlapping epitopes (Fig. 2f). We postulated that a bi-functional IgG ReconnAb containing both CV10 and COV2-2449 would make viral escape even less likely, because it could bind to two distinct conserved epitopes. To produce a bi-functional IgG ReconnAb, we applied the clinically used 41 CrossMAb platform 42 and tethered ACE2 to the LC of only one of the IgG arms (Fig. 4b). This allows, as with the scFv-ACE2 fusions, a stoichiometry of only a single ACE2 per ReconnAb, such that ACE2 remains monovalent before and after TEV cleavage.
We expressed and purified the CV10-2449-ACE2 CrossMAb (Extended Data Fig. 7a) and found that it bound to SARS-CoV-2 as expected (Extended Data Fig. 7b). As well, the uncleaved CrossMAb competed substantially with ACE2 (Extended Data Fig. 8a) and showed binding to FcγRI (Extended Data Fig. 8b). Finally, dependent on the presence of the tether, the CV10-2449-ACE2 CrossMAb neutralized all SARS-CoV-2 VOCs, including Omicron, at sub-nanomolar concentrations (Fig. 4c). Moreover, we found that our IgG ReconnAb was able to neutralize the BA.2 variant of Omicron (Fig. 4d). The neutralization potency generated by our IgG ReconnAb, with a monovalent ACE2, was substantially more robust than that generated by bivalent ACE2 (Fc-ACE2) alone (Extended Data Fig. 9), suggesting that the non-neutralizing binding component is conferring more benefit than an additional, low-affinity neutralizing component. These   results were similarly reflected in a limiting dilution live viral neutralization assay (Supplementary Table 1). Taken together, the results described here lay a foundation for the development of ReconnAbs as a novel class of broadly neutralizing therapeutics.

Discussion
Traditionally, the discovery of therapeutic biologics against infectious diseases has focused on identifying agents with neutralizing activity. We demonstrate here using ReconnAbs that cross-reactive, non-neutralizing antibodies, which have been often largely overlooked, can be powerful reagents in the creation of potent, broad-spectrum anti-viral agents.
ReconnAbs have two main components: a binding component, the non-neutralizing antibody that binds with high affinity to a conserved region on the spike protein, and an inhibitory component, in our case the ACE2 domain 43 . Because therapeutics containing ACE2 run the risk of eliciting autoimmunity in humans, our use of ACE2 as the inhibitory component represents a proof of concept of the ReconnAb design. The ACE2 module could be replaced by other neutralizing components, such as ACE2 domains with enhanced RBD-binding activity 44,45 , aptamers 46 or RBD-directed mAbs 1,[36][37][38]47 . It is noteworthy that we observed broad-spectrum efficacy with our ReconnAbs using monovalent ACE2, which, by itself, is weakly neutralizing; this suggests that, if a ReconnAb were made using a high-affinity RBD-directed antibody, efficacy would be sustained even if RBD escape mutations decreased affinity substantially. Future ReconnAb designs could also target the interaction with dipeptidyl peptidase 4 (DPP4), a receptor for other coronaviruses 48   Improvements could also be made to the conserved, nonneutralizing antibodies. Our library of SARS-CoV-2 non-RBD antibodies was derived from sequences early in the COVID-19 pandemic, which is relatively small in scope. The library does not contain, for instance, any vaccine-derived antibodies, which are known to include cross-reactive, non-neutralizing antibodies 25 . Future iterations of this work could start with much larger libraries 33 , with the potential to identify antibodies and/or nanobodies that target the most highly conserved epitopes and that are least likely to undergo mutational escape 49 . Although we have focused on non-neutralizing antibodies, neutralizing antibodies that bind to conserved epitopes might also be useful as the conserved component of ReconnAbs. Other features, such as linkage locations and length, fusion partners and modifications to the Fc domains, can be tuned in subsequent ReconnAb designs and will likely play an important role in their future conversion to therapeutics 50 .
Compared to neutralizing epitopes, highly conserved, nonneutralizing epitopes are less likely, in theory, to be subject to immune pressure, because antibody binding at these sites does not affect the ability of the virus to infect cells. Omicron provides strong evidence that SARS-CoV-2 viral evolution responds to immune pressure by mutating neutralizing epitopes ( Fig. 1) 2,3,16 . ReconnAbs demonstrate the powerful utility of a non-active component, if it targets a highly conserved epitope, in the development of therapeutics. Indeed, we consider ReconnAbs to be a considerably more viable long-term option for SARS-CoV-2 therapy than the current standard where new monoclonals will need to be developed-for example, bebtelovimab 13 for BA.2-with new emerging variants.
Finally, we anticipate that interrogation of existing antibody libraries for highly conserved, non-neutralizing binders will facilitate production of ReconnAbs, not just for SARS-CoV-2 but also for other viruses, such as HIV-1, influenza or other hCoVs. We see  ReconnAbs as having utility not only in the current pandemic but also in mitigating the impact of future pandemics. Strategic stockpiles of customized ReconnAbs and rapid administration in a pandemic setting could alleviate the initial impact of a new pathogen, allowing time for other therapeutics and countermeasures to be put into place.

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scFv design. The sequences of these 48 antibodies were then converted into scFv sequences by linking the HC variable region to the LC variable region with a G4S-3 linker (GGGGSGGGGSGGGGS). All scFvs were designed in the following order: signal sequence-HC-G4S-3-LC. This vector also contained the HVM06_Mouse Ig HC V region 102 signal peptide (MGWSCIILFLVATATGVHS) to allow for protein secretion and purification from the supernatant. After construct design, the plasmids were ordered with the sequences inserted at the XhoI and NheI sites in the pTwist CMV BetaGlobin vector (Twist Biosciences).
Library production. ScFvs were produced as Aga2p fusions 57 . In brief, 4 µg of pPNL6 vector in Cut Smart buffer was digested using 1 µl of NheI HF and BamHI HF (New England Biolabs) at 37 °C for 1 hour. Digested plasmid was then gel extracted using the Thermo Fisher Scientific Gel Extraction Kit. Equimolar aliquots of each scFv plasmid were pooled, and the resultant pool was amplified using primers that annealed to the hexa-his tag (reverse primer) or signal peptide (forward primer) and had a 50-bp overlap with the pPNL6 vector digested with NheI and BamHI. The pooled amplification was gel extracted to ensure that it was the correct size. Yeast were prepared by first streaking a YPAD plate and incubating for 2-3 days until single colonies were identifiable. A single colony was inoculated in 5 ml of YPAD with overnight shaking at 30 °C. Cultures were harvested into six tubes and pelleted. Yeast were resuspended in electroporation buffer (10 mM Tris base, 250 mM sucrose, 2 mM MgCl 2 ) containing the gel-extracted library amplification and digested pPNL6 vector. This mixture was then pulsed, and the electroporated yeast were recovered in SD-CAA media overnight (30 °C shaking). These yeast were then induced by a 1:10 dilution into SG-CAA media and grown at 20 °C shaking for 2-3 days.
Yeast binding. After induction in SG-CAA shaking for 2-3 days at 20 °C, the yeast library, expressing surface-exposed scFvs, was incubated for 15 minutes with a dilution of pre-formed baits. Baits were formed by mixing biotinylated baits and streptavidin 647 (Jackson ImmunoResearch) at a 4:1 ratio.  . Cells were sorted directly into tubes containing 4 ml of SD-CAA media. These sorted libraries were grown for 1 day at 30 °C shaking in SD-CAA media, and then 300 µl of the cultures were mini-prepped (Zymo Research), following the manufacturer's protocol. Mini-prepped DNA was transformed into STELLAR Competent Cells (Clontech) and plated on carbenicillin LB agar plates (as per pPNL6ʼs resistance marker). Escherichia coli cells that grow should, theoretically, contain only a single sequence from each of the yeast that were sorted above. Ten E. coli colonies from the hi-gate and 20 E. coli colonies from the low-gate sort were sent for sequencing (Sequetech). The sequences were then analyzed by sequence alignment using SnapGene software (version 6.0.2).
Constructs. scFv-ACE2 fusion proteins. scFvs identified as cross-reacting with SARS-CoV-1 and falling into a unique epitope (CV10, CV27, COVA2-14, COV2-2449 and COV2-2143) sort were cloned into the pTwist CMV BetaGlobin vector such that they contained a l in ker ( GG SG SH HH HH HA ST GG GS GG PS GQ AG-AA AS EE NL YF QG SLFVSNHAYGGSGGEARV), followed by the ectodomain of human ACE2.
LC and LC-ACE2 fusion proteins. Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter. The antibodies variable LC were cloned between the CMV promoter and the bGH poly(A) signal sequence of the CMV/R plasmid to facilitate improved protein expression. The variable region was cloned into the human IgG1 backbone with a kappa LC. This vector also contained the HVM06_Mouse (P01750) Ig HC V region 102 signal peptide to allow for protein secretion and purification from the supernatant. The LCs from the scFvs from the above-described SARS-CoV-1 sort were cloned into the CMV/R vector in-frame with the kappa LC. For COV2-2449, the LC was additionally cloned such that there was a C-terminal linker ( GGSGSHHHHHHAS TGGGSGGPSGQAGAAASEENLYFQGSLFVSNHAYGGSGGEARV), followed by the ectodomain of human ACE2.
HC IgG plasmids. Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter. The antibodies variable HC were cloned between the CMV promoter and the bGH poly(A) signal sequence of the CMV/R plasmid to facilitate improved protein expression. The variable region was cloned into the human IgG1 backbone. This vector also contained the HVM06_Mouse (P01750) Ig HC V region 102 signal peptide to allow for protein secretion and purification from the supernatant. The HCs from the scFvs from the above-described SARS-CoV-1 sort were cloned into the CMV/R vector in-frame with HC constant regions.
hCoV spike protein constructs. Spike proteins from six hCoVs were cloned into a pADD2 vector between the rBeta-globin intron and β-globin poly(A). A total of 48 constructs were cloned and tested containing a C-terminal truncation or not, a T4 foldon or GCN4 trimerization domain and an Avi tag or not. Each set of eight proteins was produced for the six hCoV spike proteins from SARS-CoV-2, SARS-CoV-1, MERS, 229E, NL63 and OC43. Depictions of the constructs and linkers produced are shown in Extended Data Fig. 2.
DNA preps. The 48 spike protein constructs from the hCoVs were mirA-prepped 59 using Thermo Fisher Scientific GeneJET plasmid mini-prep kit. Eight milliliters of E. coli containing the constructs were harvested by centrifugation, and 200 µl of freshly made resuspension buffer was added to each clone. Then, 200 µl of lysis buffer was added, followed by inversion, and then 300 µl of neutralization buffer was added. Lysed E. coli was then centrifuged at >18,000g for 10 minutes. The supernatant was transferred to a tube containing 580 µl of 100% EtOH. The EtOH solution was then added to a GeneJET plasmid mini-prep column, and the regular wash steps and elution steps were followed. mirA-preps resulted in significantly more plasmid production and allowed for small-scale transfection of the 48 clones tested. For the FL-GCN4-Avi-His expression tests and protein production, all samples were maxi-prepped from 200 ml of E. coli using NuceloBond Xtra Maxi Kit per the manufacturer's recommendations (Macherey-Nagel). All scFv-ACE2, CrossMAb 60 , antibody, hFc-ACE2 and lentiviral plasmids were maxi-prepped in the same fashion.
Protein production. All proteins were expressed in Expi293F cells. Expi293F cells were cultured in media containing 66% Freestyle/33% Expi media (Thermo Fisher Scientific) and grown in TriForest polycarbonate shaking flasks at 37 °C in 8% CO 2 .
The day before transfection, cells were spun down and resuspended to a density of 3 × 10 6 cells per ml in fresh media. The next day, cells were diluted and transfected at a density of approximately 3-4 × 10 6 cells per ml. Transfection mixtures were made by adding the following components: mirA-prepped or maxi-prepped DNA, culture media and FectoPro (Polyplus) would be added to cells to a ratio of 0.5-0.8 µg:100 µl:1.3 µl:900 µl. For example, for a 100-ml transfection, 50-80 µg of DNA would be added to 10 ml of culture media, and then 130 µl of FectoPro would be added to this. After mixing and a 10-minute incubation, the resultant transfection cocktail would be added to 90 ml of cells. The cells were harvested 3-5 days after transfection by spinning the cultures at >7,000g for 15 minutes. Supernatants were filtered using a 0.22-µm filter. To determine hCoV protein expression, spun-down Expi293F supernatant was used without further purification. For proteins containing a biotinylation tag (Avi-Tag), Expi293F cells containing a stable BirA enzyme insertion were used, resulting in spontaneous biotinylation during protein expression.
Protein purification-Fc Tag-containing proteins. All proteins containing an Fc tag (for example, IgGs, CrossMAb-Ace2 fusions and hFc-ACE2) were purified using a 5-ml MAb Select SuRe PRISM column on the ÄKTA pure fast protein liquid chromatography (FPLC) system (Cytiva). Filtered cell supernatants were diluted with 1/10 volume of 10× PBS. The ÄKTA system was equilibrated with: A1 -1× PBS; A2 -100 mM glycine pH 2.8; B1 -0.5 M NaOH; Buffer line -1× PBS; and Sample lines -H 2 O. The protocol washes the column with A1, followed by loading of the sample in Sample line 1 until air is detected in the air sensor of the sample pumps, followed by 5 column volume washes with A1 and elution of the sample by flowing of 20 ml of A2 (directly into a 50-ml conical containing 2 ml of 1 M Tris pH 8.0), followed by 5 column volumes of A1, B1 and A1. The resultant Fc-containing samples were concentrated using 50-kDa or 100-kDa cutoff centrifugal concentrators. Proteins were buffer exchanged using a PD-10 column (Sephadex) that had been pre-equilibrated into 20 mM HEPES and 150 mM NaCl. IgGs used for competition, binding and neutralization experiments were not further purified. CrossMAb-ACE2 fusions were then further purified using the S6 column on the ÄKTA system.
Protein purification-His-tagged proteins. All proteins not containing an Fc tag (for example, scFvs and scFv fusions and FL spike trimers from hCoV polypeptide antigens) were purified using HisPur Ni-NTA resin (Thermo Fisher Scientific). Cell supernatants were diluted with 1/3 volume of wash buffer (20 mM imidazole, 20 mM HEPES pH 7.4 and 150 mM NaCl), and the Ni-NTA resin was added to diluted cell supernatants. For all mixtures not containing hCoV spike protein, the samples were then incubated at 4 °C while stirring overnight. hCoV spike proteins were incubated at room temperature. Resin-supernatant mixtures were added to chromatography columns for gravity flow purification. The resin in the column was washed with wash buffer (20 mM imidazole, 20 mM HEPES pH 7.4 and 150 mM NaCl), and the proteins were eluted with 250 mM imidazole, 20 mM HEPES pH 7.4 and 150 mM NaCl. Column elutions were concentrated using centrifugal concentrators (50-kDa cutoff for scFv-ACE2 fusions and 100-kDa cutoff for trimer constructs), followed by size-exclusion chromatography on an ÄKTA pure system. ÄKTA pure FPLC with a Superdex 6 Increase gel filtration column (S6) was used for purification. Then, 1 ml of sample was injected using a 2-ml loop and run over the S6, which had been pre-equilibrated in de-gassed 20 mM HEPES and 150 mM NaCl before use. Biotinylated antigens were not purified using the ÄKTA pure system. . hCoV supernatants were assessed for binding using Anti-Penta His (His1K) tips. These tips are designed to bind specifically to a Penta-His tag on proteins. For this experiment, tips were baselined in a blank well and then associated in the wells containing 50 µl of hCoV expression media and 150 µl of Octet buffer. Response values (that is, peak reached after 5 minutes of association) were determined using the Octet data analysis software. Final data analysis was done in Prism. BLI (Octet) binding experiments-IgG binding. All reactions were run on an Octet RED96, and samples were run in PBS with 0.1% BSA and 0.05% Tween 20 (Octet buffer). IgGs produced from the scFvs from the above sort were assessed for binding using streptavidin biosensors (Sartorius/ForteBio) loaded to a threshold of 0.8 nm of SARS-CoV-2, SARS-CoV-1, MERS and OC43 biotinylated spike proteins. Tips were then washed and baselined in wells containing only Octet buffer. Samples were then associated in wells containing 100 nM IgG. A control well that loaded antigen but associated in a well containing only 200 µl of Octet buffer was used as a baseline subtraction for data analysis.
BLI (Octet) binding experiments-IgG competition. All reactions were run on an Octet RED96, and samples were run in PBS with 0.1% BSA and 0.05% Tween 20 (Octet buffer). IgGs produced from the scFvs from the above sort were assessed for their competition of binding with one another using Anti-Penta His (His1K) biosensors (Sartorius/ForteBio). His1K tips were pre-quenched with buffer containing 10 nM biotin. Tips were then loaded with 100 nM protein for 2 minutes (SARS-CoV-2 spike) or 4 minutes (SARS-CoV-1 spike). These tips were then associated with one of seven antibodies (CV27, COV2-2147, CV10, COVA2-14, COVA2-18, COV2-2449 or COV2-2143) at 100 nM for 5 minutes to reach saturation. Tips were baselined and then associated with one of the seven antibodies. For this step, all eight tips went into the same antibody at 100 nM. Response values (that is, peak reached after 2 minutes of association) was determined using Octet data analysis software. Values were normalized to the tip loaded with either SARS-CoV-2 or SARS-CoV-1 spike but without a competing antibody. These values were set as a value of 1 for each antibody. This is simply the antibody binding to the protein. Additionally, the antibody competing with itself was set to a value of 0. Final data analysis was done in Prism. BLI (Octet) binding experiments-scFv-ACE2 fusion. All reactions were run on an Octet RED96, and samples were run in PBS with 0.1% BSA and 0.05% Tween 20. Streptavidin biosensors (Sartorius/ForteBio) were loaded for 2 minutes with 100 nM biotinylated antigens (SARS-CoV-2 or SARS-CoV-1 spike proteins). Samples were then washed and baselined in wells containing Octet buffer. Association occurred in samples containing ACE2 fusion proteins either without or with TEV protease (New England Biolabs) treatment. scFv-ACE2 fusions were tested at 200 nM. Association was conducted for 2 minutes, and dissociation was conducted for 1 minute. BLI (Octet) binding experiments-scFv-ACE2 fusion and CrossMAb-ACE2 fusion competition with hFc-ACE2 (ref. 61 ). All reactions were run on an Octet RED96, and samples were run in PBS with 0.1% BSA and 0.05% Tween 20 (Octet buffer). Streptavidin biosensors (Sartorius/ForteBio) (scFvs) or His1K biosensors (Sartorius/ForteBio) (CrossMAb) were loaded for 2 minutes with 100 nM biotinylated antigens (SARS-CoV-2 or SARS-CoV-1 spike-scFvs) or 4 minutes with 200 nM His-tagged antigens (CrossMAb). Samples were then washed and baselined in wells containing Octet buffer. scFv-ACE2 fusions or CrossMAbs were then associated for 5 minutes. Samples were baselined and then associated with either hFc-ACE2 for 2 minutes (scFv) or 40 seconds (CrossMAb). Response values was determined using Octet data analysis software. Samples that loaded SARS-CoV-2 or SARS-CoV-1 but did not associate with any hFc-ACE2 were used as a baseline subtraction. Values were normalized to the binding of hFc-ACE2 without a competitor.