Sybodies targeting the SARS-CoV-2 receptor-binding domain

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has resulted in a global health and economic crisis of unprecedented scale. The high transmissibility of SARS-CoV-2, combined with a lack of population immunity and prevalence of severe clinical outcomes, urges the rapid development of effective therapeutic countermeasures. Here, we report the generation of synthetic nanobodies, known as sybodies, against the receptor-binding domain (RBD) of SARS-CoV-2. In an expeditious process taking only twelve working days, sybodies were selected entirely in vitro from three large combinatorial libraries, using ribosome and phage display. We obtained six strongly enriched sybody pools against the isolated RBD and identified 63 unique anti-RBD sybodies which also interact in the context of the full-length SARS-CoV-2 spike ectodomain. Among the selected sybodies, six were found to bind to the viral spike with double-digit nanomolar affinity, and five of these also showed substantial inhibition of RBD interaction with human angiotensin-converting enzyme 2 (ACE2). Additionally, we identified a pair of anti-RBD sybodies that can simultaneously bind to the RBD. It is anticipated that compact binders such as these sybodies could feasibly be developed into an inhalable drug that can be used as a convenient prophylaxis against COVID-19. Moreover, generation of polyvalent antivirals, via fusion of anti-RBD sybodies to additional small binders recognizing secondary epitopes, could enhance the therapeutic potential and guard against escape mutants. We present full sequence information and detailed protocols for the identified sybodies, as a freely accessible resource.


INTRODUCTION
The ongoing pandemic arising from the emergence of the 2019 novel coronavirus, SARS-CoV-2, demands urgent development of effective antiviral therapeutics. Several factors contribute to the adverse nature of SARS-CoV-2 from a global health perspective, including the absence of herd immunity [1], high transmissibility [2,3], the prospect of asymptomatic carriers [4], and a high rate of clinically severe outcomes [5]. Moreover, a vaccine against SARS-CoV-2 is unlikely to be available for at least 12-18 months [6], despite earnest development efforts [7,8], making alternative intervention strategies paramount. In addition to offering relief for patients suffering from the resulting COVID-19 disease, therapeutics may also reduce the viral transmission rate by being administered to asymptomatic individuals subsequent to probable exposure [9]. Finally, given that SARS-CoV-2 represents the third global coronavirus outbreak in the past 20 years [10,11], development of rapid therapeutic strategies during the current crises could offer greater preparedness for future pandemics.
Akin to all coronaviruses, the viral envelope of SARS-CoV-2 harbors protruding, club-like, multidomain spike proteins that provide the machinery enabling entry into human cells [12][13][14]. The spike ectodomain is segregated into two regions, termed S1 and S2. The outer S1 subunit of SARS-CoV-2 is responsible for host recognition via interaction between its C-terminal receptor-binding domain (RBD) and human angiotensin converting enzyme 2 (ACE2), present on the exterior surface of airway cells [14,15]. While there is no known host-recognition role for the S1 N-terminal domain (NTD) of SARS-CoV-2, it is notable that S1 NTDs of other coronaviruses have been shown to bind host surface glycans [12,16]. In contrast to spike region S1, the S2 subunit contains the membrane fusion apparatus, and also mediates trimerization of the ectodomain [12][13][14]. Prior to host recognition, spike proteins exist in a metastable pre-fusion state wherein the S1 subunits lay atop the S2 region and the RBD oscillates between "up" and "down" conformations that are, respectively, accessible and inaccessible to receptor binding [12,17,18]. Upon processing at the S1/S2 and S2' cleavage sites by host proteases as well as engagement to the receptor, the S2 subunit undergoes dramatic conformational changes from the pre-fusion to the post-fusion state. Such structural rearrangements are associated with the merging of the viral envelope with host membranes, thereby allowing injection of the genetic information into the cytoplasm of the host cell [19,20].
Coronavirus spike proteins are highly immunogenic [21], and several experimental approaches have sought to target this molecular feature for the purpose of viral neutralization [22]. The high specificity, potency, and modular nature of antibody-based antiviral therapeutics has shown exceptional promise [23][24][25], and the isolated, purified RBD has been a popular target for the development of anti-spike antibodies against pathogenic coronaviruses [26][27][28][29]. However, binders against the isolated RBD may not effectively engage the aforementioned pre-fusion conformation of the full spike, which could account for the poor neutralization ability of recently described single-domain antibodies that were raised against the RBD of SARS-CoV-2 [30]. Therefore, to better identify molecules with qualities befitting a drug-like candidate, it would be advantageous to validate RBD-specific binders in the context of the full, stabilized, pre-fusion spike assembly [13,31].
Single domain antibodies based on the variable VHH domain of heavy-chain-only antibodies of camelids -generally known as nanobodies -have emerged as a broadly utilized and highly successful antibody fragment format [32]. Nanobodies are small (12)(13)(14)(15), stable, and inexpensive to produce in bacteria and yeast [33], yet they bind targets in a similar affinity range as conventional antibodies. Due to their minimal size, they are particularly suited to reach hidden epitopes such as crevices of target proteins [34]. We recently designed three libraries of synthetic nanobodies, termed sybodies, based on elucidated structures of nanobody-target complexes (Fig. 1A) [35,36]. Sybodies can be selected against any target protein within twelve working days, which is considerably faster than natural nanobodies, which requires the repetitive immunization during a period of two months prior to binder selection by phage display [36] (Fig. 1C). A considerable advantage of our platform is that sybody selections are carried out under defined conditions -in case of coronavirus spike proteins, this offers the opportunity to generate binders recognizing the metastable pre-fusion conformation [13,14]. Finally, due to the feasibility of inhaled therapeutic nanobody formulations [37], virusneutralizing sybodies could offer a convenient and direct means of prophylaxis.
Here, we report of in vitro selection and characterization of sybodies against the RBD of SARS-CoV-2 spike protein. Two independently prepared RBD constructs were used for in vitro sybody selections, and resulting single clones that could bind the full spike ectodomain were sequenced, expressed, and purified. Six unique sybodies show favorable binding affinity to the SARS-CoV-2 spike, and five of these were also found to substantially attenuate the interaction between the viral RBD and human ACE2. Moreover, pairs of sybodies were identified that can simultaneously bind to the RBD. We present all sequences for these clones, along with detailed protocols to enable the community to freely produce and further characterize these SARS-CoV-2 binders.

Purification and biotinylation of target proteins
Based on sequence alignments with isolated RBD variants from SARS-CoV-1 that were amenable to purification and crystallization [29,38], a SARS-CoV-2 RBD construct was designed, consisting of residues Pro330-Gly526 fused to Venus YFP (RBD-vYFP). This construct was expressed and secreted from Expi293 cells, and RBD-vYFP was extracted directly from culture medium supernatant using an immobilized anti-GFP nanobody [39], affording a highly purified product with negligible background contamination. Initial efforts to cleave the C-terminal vYFP fusion partner with 3C protease resulted in unstable RBD, so experiments were continued with full RBD-vYFP fusion protein. To account for the presence of the vYFP fusion partner, a second RBD construct, consisting of a fusion to murine IgG1 Fc domain (RBD-Fc), was commercially acquired. To remove any trace amines, buffers were exchanged to PBS via extensive dialysis. Proteins were chemically biotinylated, and the degree of biotinylation was assessed by a streptavidin gel-shift assay and found to be greater than 90 % of the target proteins [40]. We note that while both RBD fusion proteins were well-behaved, a commercially acquired purified full-length SARS-CoV-2 spike ectodomain construct (ECD) was found to be aggregation-prone. Very recently, we also produced an engineered spike protein ectodomain containing two point mutations known to stabilize the pre-fusion state, an inactivated furin cleavage site, and a C-terminal trimerization motif [13,14,31]. While this purified pre-fusion spike (PFS) had not yet been available for binder selections and characterization by grating-coupled interferometry, it was used to conduct ELISAs in order to identify selected sybodies which recognize the RBD in the pre-fusion context (see below).

Sybody selections
Since both our RBD constructs bear additional fusion domains (Fc of mouse IgG1 and vYFP, respectively), sybody selections were carried out with a "target swap" approach (Fig. 1B). Hence, selections with the three sybody libraries (concave, loop and convex) were started with the RBD-vYFP construct using ribosome display, and the RBD-Fc construct was then used for the two phage display rounds (selection variant 1: RBD-vYFP/RBD-Fc/RBD-Fc) and vice versa (selection variant 2: RBD-Fc/RBD-vYFP/RBD-vYFP). Accordingly, there were a total of six selection reactions (Table 1, Fig. 1B). To increase the average affinity of the isolated sybodies, we included an off-rate selection step using the preenriched purified sybody pool after phage display round 1 as competitor. To this end, sybody pools of all three libraries of the same selection variant were sub-cloned from the phage display vectors into the sybody expression vector pSb_init. Subsequently, the two separate pools (all sybodies of selection variants 1 and 2, respectively) were expressed and purified. The purified pools were then added to the panning reactions of the respective selection variant in the second phage display round. Thereby, rebinding of sybody-phage complexes with fast off-rates was suppressed. Enrichment of sybodies against the RBD was monitored by qPCR. Already in the first phage display round, the concave and loop sybodies of selection variant 2 showed enrichment factors of 7 and 3, respectively (Table 1). After the second phage display round (which included the off-rate selections step), strong enrichment factors in the range of 10-263 were determined.

Sybody identification by ELISA
After sub-cloning the pools from the phage display vector pDX_init into the sybody expression vector pSb_init, 47 clones of each of the 6 selections reactions ( Table 1, Fig. 1B) were picked at random and expressed in small scale. Our standard ELISA was initially performed using RBD-vYFP (RBD), spike ectodomain containing S1 and S2 (ECD), and maltose binding protein (MBP) as unrelated dummy protein. As outlined in the Materials and Methods section, ELISA analysis revealed very high hit rates for the RBD and the ECD ranging from 81 % to 100 % and 66 % to 96 %, respectively (Fig. 2, Table 1). The majority of the sybodies giving an ELISA signal to the RBD also gave a clear signal the full-length spike protein (Fig. 2). However, there was a total of 44 hits that only gave an ELISA signal for RBD-vYFP, but not for the ECD. This could be due to the presence of cryptic RBD epitopes that are not accessible in the context of the full-length spike protein, or the respective sybodies may recognize the vYFP portion of the RBD-vYFP construct, though the selection procedure clearly disfavors the latter explanation. Importantly, background binding to the dummy protein MBP was not observed for any of the analyzed sybodies, clearly showing that the binders are highly specific. We then sequenced 72 sybodies that were ELISA-positive against RBD-vYFP as well as the full-length spike (12 for each of the 6 selection reactions numbered from Sb#1-72, see also Fig. 1B).
Subsequent to sybody sequencing, we also performed the ELISA using engineered pre-fusion-stabilized spike ectodomain (PFS) (Fig. 2), which was not available at the onset of the project. Overall, the ELISA signals for the ECD and PFS are highly similar. However, there are around 40 sybodies that bind to the ECD clearly stronger than to the PFS (yet the opposite scenario was never observed). This could be explained by the fact that the PFS forms a trimer, while the oligomeric state of the ECD is not clear. In addition, the ECD might adopt partially or completely a post-fusion state, whereas PFS is expected to predominantly adopt the pre-fusion state. Trimer formation as well as pre-fusion stabilization might shield certain binding epitopes on the RBD in the context of the PFS, which might become accessible as the spike falls apart into monomers and/or transits to the post-fusion state. In light of our ELISA data, the PFS construct will be a crucial element in any future sybody selection campaigns.

Sequence analysis
Sequencing results of 70 out of 72 sybody clones were unambiguous. Out of these 70 clones, 63 were found to be unique and the respective clone names are indicated in the ELISA figure (Fig. 2, Table 2). Of note, there were no duplicate binders identified in both selection variants, indicating that the two separate selection streams gave rise to completely different arrays of sybodies. As an additional note, one sybody identified from the supposed convex library turned out to belong to the concave library; spill-over of sybodies across libraries is occasionally observed. Hence, there was a total of 23 concave, 22 loop and 18 convex sybodies, which were then aligned according to their library origin . As a final analysis, all sybody sequences were aligned to generate a phylogenetic tree, which shows a clear segregation across the three libraries and indicates a large sequence variability of the identified sybodies ( Fig. 6).

Purification of sybodies and kinetic analysis of sybody interaction with SARS-CoV-2 proteins
The 63 unique sybodies were individually expressed in E. coli and purified via Ni-NTA affinity chromatography and gel filtration. Ultimately, 57 sybodies were sufficiently well-behaved, with respect to solubility, yield, and monodispersity, to proceed with further characterization. For a kinetic analysis of sybody interactions with the viral spike, we employed grating-coupled interferometry (GCI) to probe sybody binding to immobilized RBD-vYFP or ECD. First, the 57 purified sybodies were subjected to an off-rate screen, which revealed six sybodies (Sb#14, Sb#15, Sb#16, Sb#42, Sb#45, and Sb#68) with strong binding signals and comparatively slow off-rates. Binding constants were then determined by measuring on-and off-rates over a range of sybody concentrations, revealing affinities within a range of 20-180 nM to the SARS-CoV-2 spike (Fig. 7, Table 3). Of note, binding affinities were consistently equal or higher for the ECD as compared to the RBD-vYFP, in particular in case of Sb#68 for which the off-rate differs by more than two-fold. This might indicate a binding avidity effect arising from binding epitopes clustering in the context of the spike trimer or differences with regards to the glycan structures (RBD-vYFP was produced in HEK cells, whereas the ECD was produced in insect cells). To our surprise, the majority of purified and ELISA-positive sybodies (51 out of 57) displayed binding affinities worse than 200 nM. This may be attributed to the presence of complex heterogeneous Asnlinked glycans within the RBD, which could hinder the isolation of specific high-affinity binders. Alternatively, given that the final ELISA step of the selection process resulted in a substantial number of positive clones, insufficiently stringent conditions may have favored the high positive hit rate of lowaffinity binders.

ACE2 competition analysis
Since virulence of SARS-CoV-2 is dependent on the ability of the viral RBD to bind to human ACE2 (hACE2), we sought to determine which of the 57 selected sybodies that were well-behaved upon purification could inhibit interaction between the isolated RBD and purified hACE2. For this assessment, ELISA plates were coated with purified hACE2, and the binding of purified RBD to the 6 immobilized hACE2 was measured in the presence or absence of an excess of each purified sybody (Fig. 8). While the absence of any added sybody resulted in a strong ELISA signal corresponding to RBD association with hACE2, the pre-incubation of nearly all sybodies with the RBD resulted in an attenuated signal, implying that these binders inhibit RBD-hACE2 association. This signal decrease relative to unchallenged RBD was modest for most sybodies, with an average signal reduction of about 50%, but five sybodies demonstrated exceptionally high apparent inhibition of RBD-hACE2 interaction (Sb#14, Sb#15, Sb#16, Sb#42, and Sb#45), showing ≥90% signal reduction. Notably, the aforementioned kinetic analysis had shown that these sybodies were also among the strongest RBD binders. Taken together, this data suggests that Sb#14, Sb#15, Sb#16, Sb#42, and Sb#45 recognize a surface region on the RBD that overlaps with the hACE2 binding site.

Simultaneous binding of multiple sybodies to the RBD
While kinetic analysis had revealed Sb#68 to be among the stronger binders to the SARS-CoV-2 ectodomain (KD ≈ 37 nM, Fig. 7, Table 3), the hACE2 competition ELISA revealed that Sb#68 does not inhibit hACE2-RBD interaction to the same extent as other sybodies with comparable affinities (65% inhibition for Sb#68, compared to >90% for Sb#14, Sb#15, Sb#16, and Sb#45). Therefore, it was hypothesized that Sb#68 may interact with a non-or partially-overlapping surface on the RBD, relative to the more strongly-inhibiting sybodies. Using Sb#15 as a representative of the hACE2-inhibiting sybodies, we analyzed the ability of Sb#15 and Sb#68 to simultaneously associate with the RBD. First, ELISA experiments demonstrate that incubation of Sb#68 with the pre-fusion spike only slightly prevents the spike from binding to immobilized Sb#15, whereas pre-incubation with Sb#14, Sb#15, Sb#16, Sb#42, or Sb#45 completely prevents spike interaction with immobilized Sb#15 (Fig. 9). In agreement with the ELISA data, GCI experiments revealed that co-injection of Sb#15 and Sb#68 results in a clear (but not fully additive) increase of the response signal, relative to Sb#15 or Sb#68 injected alone, implying simultaneous binding of Sb#15 and Sb#68 (Fig. 9). The control GCI experiment involving the co-injection of Sb#15 and Sb#45 did not result in a similar signal increase ( Fig. 9). In sum, this data plausibly suggests that Sb#15 and Sb#68 can simultaneously bind to the RBD. For the design of therapeutics against SARS-CoV-2, the fusion of such a pair of non-overlapping binders could provide benefits via increased overall avidity to the spike protein.

Conclusion and outlook
We have demonstrated the ability of our rapid in vitro selection platform to generate sybodies against the SARS-CoV-2 RBD, within a two-week timeframe. Characterization of these sybodies has identified a high-affinity subset of binders that also inhibit the RBD-ACE2 interaction. We anticipate that the presented panel of anti-RBD sybodies could be of use in the design of urgently required therapeutics to mitigate the COVID-19 pandemic, particularly in the development of inhalable prophylactic formulations [37]. Furthermore, our identification of a pair of sybodies that can simultaneously associate with the RBD may offer an attractive foundation for the construction of a polyvalent sybodybased therapeutic. We have attempted to provide a complete account of the generation of these molecules, including full sequences and detailed methods, such that other researchers may contribute to their ongoing analysis. Future work may include virus neutralization assays using the identified sybodies, as well as further selection campaigns targeting additional spike epitopes. Finally, our recently described flycode technology could be utilized for deeper interrogation of selection pools, in order to facilitate discovery of exceptional sybodies that possess very slow off-rates or recognize rare epitopes [41].

Biotinylation of target proteins
To remove amines, all proteins were first extensively dialyzed against RBD buffer. Proteins were concentrated to 25 µM using Amicon Ultra concentrator units with a molecular weight cutoff of 30 -8 50 kDa. Subsequently, the proteins were chemically biotinylated for 30 min at 25°C using NHS-Biotin (Thermo Fisher, #20217) added at a 10-fold molar excess over target protein. Immediately after, the three samples were dialyzed against TBS pH 7.5. During these processes (first dialysis/ concentrating/ biotinylation/ second dialysis), 20 %, 30 %, 65 % and 44% of the RBD-vYFP, RBD-Fc, ECD and PFS respectively were lost due to sticking to the concentrator filter or due to aggregation. Biotinylated RBD-vYFP, RBD-Fc and ECD were diluted to 5 µM in TBS pH 7.5, 10 % glycerol and stored in small aliquots at -80°C. Biotinylated PFS was stored at 4°C in TBS pH 7.5.

Sybody selections
Sybody selections with the three sybody libraries concave, loop and convex were carried out as described in detail before [36]. In short, one round of ribosome display followed by two rounds of phage display were carried out. Binders were selected against two different constructs of the SARS-CoV-2 RBD; an RBD-vYFP fusion and an RBD-Fc fusion. MBP was used as background control to determine the enrichment score by qPCR [36]. In order to avoid enrichment of binders against the fusion proteins (YFP and Fc), we switched the two targets after ribosome display (Fig. 1B). For the offrate selections we did not use non-biotinylated target proteins as described in the standard protocol, because we did not have enough purified protein at hand to do so. Instead we sub-cloned all three libraries for both selections after the first round of phage display into the pSb_init vector (10 8 clones) and expressed the six pools in E. coli MC1061 cells. Then the pools corresponding to the same selection were pooled for purification. The two final pools were purified by Ni-NTA resin using gravity flow columns, followed by buffer exchange of the main peak fraction using a desalting PD10 column in TBS pH 7.5 to remove imidazole. The pools were eluted with 3.2 ml instead of 3.5 ml TBS pH 7.5 in order to ensure complete buffer exchange. These two purified pools were used for the off-rate selection in the second round of phage display at concentrations of approximately 390 µM for selection variant 1 (RBP-Fc) and 450 µM for selection variant 2 (RBP-YFP). The volume used for off-rate selection was 500 µl. Just before the pools were used for the off-rate selection, 0.5% BSA and 0.05% Tween-20 was added to each sample. Off-rate selections were performed for 3 minutes.

Sybody identification by ELISA
ELISAs were performed as described in detail before [36]. 47 single clones were analyzed for each library of each selection. Since the RBD-Fc construct was incompatible with our ELISA format due to the inclusion of Protein A to capture an α-myc antibody, ELISA was performed only for the RBD-vYFP (50 nM) and the ECD (25 nM) and later on with the PFS (25 nM). Of note, the three targets were analyzed in three separate ELISAs. As negative control to assess background binding of sybodies, we used biotinylated MBP (50 nM). 72 positive ELISA hits were sequenced (Microsynth, Switzerland).

Expression and Purification of sybodies
The 63 unique sybodies were expressed and purified as described [36]. In short, all 63 sybodies were expressed overnight in E.coli MC1061 cells in 50 ml cultures. The next day the sybodies were extracted from the periplasm and purified by Ni-NTA affinity chromatography (batch binding) followed by sizeexclusion chromatography using a Sepax SRT-10C SEC100 size-exclusion chromatography (SEC) column equilibrated in TBS, pH 7.5, containing 0.05% (v/v) Tween-20 (detergent was added for subsequent kinetic measurements). Six out of the 63 binders (Sb#4, Sb#7, Sb#18, Sb#34,Sb#47,Sb#61) were excluded from further analysis due to suboptimal behavior during SEC analysis (i.e. aggregation or excessive column matrix interaction).

Grating-coupled interferometry (GCI)
Kinetic characterization of sybodies binding onto SARS-CoV-2 spike proteins was performed using GCI on the WAVEsystem (Creoptix AG, Switzerland), a label-free biosensor. Biotinylated RBD-vYFP and ECD were captured onto a Streptavidin PCP-STA WAVEchip (polycarboxylate quasi-planar surface; Creoptix AG) to a density of 1300-1800 pg/mm 2 . Sybodies were first analyzed by an off-rate screen performed at a concentration of 200 nM (data not shown) to identify binders with sufficiently high affinities. The six sybodies Sb#14, Sb#15, Sb#16, Sb#42, Sb#45, and Sb#68 were then injected at increasing concentrations ranging from 1.37 nM to 1 μM (three-fold serial dilution, 7 concentrations) in TBS buffer supplemented with 0.05 % Tween-20. Sybodies were injected for 120 s at a flow rate of 30 μl/min per channel and dissociation was set to 600 s to allow the return to baseline. Sensorgrams were recorded at 25 °C and the data analyzed on the WAVEcontrol (Creoptix AG). Data were double-referenced by subtracting the signals from blank injections and from the reference channel. A Langmuir 1:1 model was used for data fitting.

Dual-sybody competition ELISA
Purified sybodies carrying a C-terminal myc-His Tag (Sb_init expression vector) were diluted to 25 nM in 100 µl PBS pH 7.4 and directly coated on Nunc MaxiSorp 96-well plates (ThermoFisher #44-2404-21) at 4°C overnight. The plates were washed once with 250 µl TBS pH 7.5 per well followed by blocking with 250 µl TBS pH 7.5 containing 0.5% (w/v) BSA per well. In parallel, chemically biotinylated prefusion Spike protein (PFS) at a concentration of 10 nM was incubated with 500 nM sybodies for 1 h at room temperature in TBS-BSA-T. The plates were washed three times with 250 µl TBS-T per well. Then, 100 µl of the PFS-sybody mixtures were added to the corresponding wells and incubated for 3 min, followed by washing three times with 250 µl TBS-T per well. 100 µl Streptavidin-peroxidase polymer (Merck, Cat#S2438) diluted 1:5000 in TBS-BSA-T was added to each well and incubated for 10 min, followed by washing three times with 250 µl TBS-T per well. Finally, to detect PFS bound to the immobilized sybodies, 100 µl ELISA developing buffer (prepared as described previously [36]) was added to each well, incubated for 1 h (due to low signal) and absorbance was measured at 650 nm. As a negative control, TBS-BSA-T devoid of protein was added to the corresponding wells instead of a PFSsybody mixture.  (47) 10 (12) 1) Sb#51 belongs to the concave library (spill-over). 2) Two sequencing reactions failed.

DATA AVAILABILITY STATEMENT
The plasmids encoding for the six highest affinity binders will very soon be available through Addgene (Addgene #153522 -#153527).  For each of the six independent selec on reac ons, 47 clones were picked at random and analyzed by ELISA. A non-randomized sybody was used as nega ve control (wells H6 and H12, respec vely). Sybodies that were sequenced are marked with the respec ve sybody name (Sb#1-72). Please note that iden cal sybodies that were found 2-3 mes are marked with the same sybody name (e.g. Sb#41). ELISA analyses shown in these graphs were performed on three different days: (1) RBD and MBP, (2) ECD, (3)  .

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Concave ADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCX-VXVGXXYXGQGTQVTVS Phylogene c tree of RBD sybodies. A radial tree was generated in CLC 8.1.3. Sybodies inhibit RBD binding to ACE2. The effect of sybodies on RBD associa on with human ACE2 was assessed with an ELISA. Individual sybodies (500 nM, sybody number shown on X-axis) were incubated with bio nylated RBD-vYFP (25 nM) and the mixtures were exposed to immobilized ACE2. Bound RBD-vYFP was detected with streptavidin-peroxidase/TMB. Each column indicates background-subtracted absorbance at 405 nm, normalized to the signal corresponding to RBD-vYFP in the absence of sybody (dashed red line).

Figure 9
Simultaneous binding of Sb#15 and Sb#68. (A) Simultaneous binding of sybodies was analyzed using gra ng-coupled interferometry on the WAVE system (Creop x AG, Switzerland). Bio nylated ECD was immobilized and the binders were injected alone and simultaneously at satura ng concentra ons (Sb#15: 200 nM, Sb#45: 500 nM, Sb#68: 500 nM). Superimposed sensorgrams are shown. (B) Compe on ELISA. Title of the graphs indicate the sybody which was directly coated on the plate at a concentra on of 25 nM. The labels on the x-axes depict the sybody used for compe on. To determine the background signal, buffer devoid of protein was added.