key: cord-0268848-tln2j5yd
authors: Custódio, Tânia F.; Das, Hrishikesh; Sheward, Daniel J; Hanke, Leo; Pazicky, Samuel; Pieprzyk, Joanna; Sorgenfrei, Michèle; Schroer, Martin; Gruzinov, Andrey; Jeffries, Cy; Graewert, Melissa; Svergun, Dmitri; Dobrev, Nikolay; Remans, Kim; Seeger, Markus A.; McInerney, Gerald M; Murrell, Ben; Hällberg, B. Martin; Löw, Christian
title: Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2
date: 2020-06-23
journal: bioRxiv
DOI: 10.1101/2020.06.23.165415
sha: 618c135eaa9266666f0e81b35f2795c258e28a89
doc_id: 268848
cord_uid: tln2j5yd

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed a novel conformation of the spike where two RBDs are in the ‘up’ ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

cryo-EM reconstruction also reveals that the spike bound to Sb23 displays a previously unobserved 92 conformation where two RBDs are in the 'up' ACE2-binding conformation.

Sybody selection, ELISA and sequencing 96 Sybody selections on biotinylated RBD were carried out with the three sybody libraries (concave, 97 loop and convex) following established procedures 26, 27 . The enrichment of binders against RBD 98 was closely followed by qPCR with enrichment factors of 100 -9000 in the last selection round, 99 indicating strong specific enrichment (Fig. 1a) . In general, sybodies that gave rise to a positive 100 ELISA signal for the RBD, did so also for the pre-fusion spike protein. ELISA signals for the spike 101 protein were on average higher compared to the RBD, which could be due to avidity effects caused indicating RBD-specificity of the selected sybodies ( Supplementary Fig. 1 ). Next, we sequenced 105 100 clones which were positive in ELISA against RBD and spike covering the three libraries 106 equally well. We obtained 85 unique sybody sequences (Supplementary Table 1 ). In total, 33 107 sybodies were derived from the concave, 32 from the loop and 20 from the convex library. Based 108 on sequence alignment and phylogenetic tree analysis (Fig. 1b) , it becomes clear that selected 109 binders cover a large sequence space. Fig. 2 ). We employed a 114 biolayer interferometry (BLI) assay to kinetically characterize the interaction of sybodies with 115 RBD and spike. Biotinylated RBD was immobilized on a streptavidin sensor and on-and off-rates 116 for all purified sybodies were determined at a single concentration (500 nM) yielding affinities 117 (KD) in a range of 5 nM -10 µM for the RBD (Supplementary Fig. 3 ). Most sybodies displayed 118 strong binding signals, but many of them exhibited fast off-rates, which is disadvantageous for 119 neutralization activity. 120 To obtain more accurate affinity data for RBD binding, we selected Sybodies 23, 42, 76 121 and 95 and determined their binding constants by analysing the on-and off-rates over a large range 122 of sybody concentrations. Resulting affinities are listed in Fig. 2a -d. Tight binding of the selected 123 binders to RBD was further confirmed by thermal shift assays (Fig. 2e, f) . In particular, the 124 interaction of Sb23 with RBD increases the melting temperature of RBD by almost ten degrees, 125 which is considerably more than for other binders. To test whether the selected sybodies can neutralize SARS-CoV-2, we performed a neutralization 129 assay with lentiviral particles pseudotyped with the SARS-CoV-2 spike protein 28 . Thirty-six 130 sybodies were screened for neutralization, identifying eleven capable of neutralizing SARS-CoV-131 2 at an IC50 <20 µg/ml, including six with an IC50 <5 µg/ml ( Supplementary Fig. 4) . No 132 neutralization of VSV-G pseudotyped viruses was evident for any sybody preparation, and a 133 control sybody targeting the human peptide transporter 2 (PepT2) did not show any neutralizing 134 activity. Sybody 23 (Sb23) represented the most potent sybody identified, with an IC50 of 0.6 135 µg/ml (Fig. 3a) . concentration range (Fig. 3b ). This could be attributed to either a direct competition for the receptor 143 binding motif, a steric hindrance or conformation induced changes upon Sb23 binding.

To confirm this observation in the context of the trimeric spike protein, we developed a 145 competition assay under equilibrium conditions to screen binding of Sb23 to the spike protein in 146 the presence or absence of ACE2 using microscale thermophoresis. This method is sensitive to 147 changes in size, charge state and hydration shell, and requires one binding partner to be 148 fluorescently labelled. We monitored binding of the fluorescently labelled Sb23 to the spike 149 protein in the presence of a constant concentration of ACE2. In case the sybody targets a different 150 epitope on the RBD of the spike protein than ACE2, the resulting affinity is expected to be 151 independent from the presence of ACE2. However, our data show that with increasing 152 concentrations of ACE2, the affinity of Sb23 to the spike protein drops 10-fold in the presence of 153 200 nM ACE2 (Fig. 3c ). In conclusion, these data indicate that Sb23 and ACE2 compete for the 154 same or overlapping binding sites on SARS-CoV-2-RBD. At the same time the data also highlight 155 that Sb23 has a significantly higher affinity to the RBD than ACE2 since Sb23 can efficiently 156 replace ACE2 from a preformed complex. To obtain a more detailed understanding, we performed SAXS-based rigid body modelling 166 of the complex between Sb23 and RBD (Fig. 4d) . The resulting hybrid rigid body model agrees 167 well with the ab initio shape of the complex (Fig. 4c ) and, importantly, in all resulting models, 168 Sb23 is placed next to the ACE2 binding site and binds sidewise to the RBD as expected for a 169 binder from the concave designed library. ACE2 binding to the 'up' protomer is hindered in the '1-up' conformation also from Sb23 binding 183 the neighbouring 'down' protomer ( Fig. 5a-c) . This interprotomer-mediated blockage is true also 184 for the corresponding 'up' protomer in the '2-up' conformation but not for the 2 nd 'up' protomer 185 in this conformation. Hence, even if the blockage of ACE2 binding in the '1-up' conformation 186 from two independent sites may contribute significantly to the efficacy in neutralization by Sb23, 187 the interprotomer-medicated blockage is reduced in the '2-up' conformation. The spike and RBD constructs were transfected and expressed according to the following 245 protocol 36 . One day before transfection, cells were seeded in culture medium at a density of 2×10 6 246 cells/ml. On the day of transfection, cells were centrifuged (5 min, RT, 100×g) and resuspended 247 in fresh FreeStyle medium to a final density of 20×10 6 cells/ml. DNA was added to a final 248 concentration of 1.5 mg/l, mixed and followed by addition of MAX PEI in 1:2 ratio (w/w).

Transfected cells were incubated at 37°C with agitation at 220 rpm in an 8% CO2 atmosphere for The binding of selected sybodies to RBD or binding of RBD to ACE2 proteins was measured by 364 biolayer interferometry (BLI) using the Octet RED96 system (FortéBio).

For the RBD binding screening assay, biotinylated RBD was loaded on streptavidin For RBD, three glycans were added to the most frequent relative abundance of glycan species, the 408 C-terminal portion was modelled as a coil, and the structure was refined using SREFLEX 46 . The 

The sybody structure was homology modelled using Phyre2 52 with PDB: 4PFE 53 as a template.

The missing regions of the RBD domains were built based on the RBD-spike crystal structure 444 (PDB: 6LZG) 32 . Structure refinement and manual model building were performed using COOT 445 and PHENIX 54 in interspersed cycles with secondary structure and geometry restrained. All unique binders identified in this study. Sequence alignment was performed using PROMAL3D 621 and the phylogenetic tree was construct using the maximum likelihood (ML) analysis in MEGA.

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Structure of the SARS-CoV-2 spike receptor-binding domain bound to the 468 ACE2 receptor

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Cryo-EM structure of the 2019-nCoV spike in the prefusion 475 conformation. Science (80-. )

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Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein 522 reveal a prerequisite conformational state for receptor binding

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Binding curves are coloured black 658 and the global fit of the data to a 1:1 binding model is red

Thermal unfolding data of isolated RBD and in complex with Sb23. Transition midpoints are 660 shown by a dashed line. (f) Resulting melting temperatures of RBD alone and in complex with 661

Sb95 and a control sybody (NC), selected against the human peptide transporter

Sb23 neutralizes SARS-CoV-2 pseudoviruses and competes with ACE2 677 (a) SARS-CoV-2 or VSV-G spike pseudotyped lentivirus was incubated with a dilution series of 678

Neutralization by Sb23 was repeated at least in 679 duplicates and the error bars represent the standard deviation. (b) BLI sensorgrams of immobilized 680

The 681 assay was performed in a concentration range of 200-12.5 nM ACE2 and fit of the data to a 1:1 682 binding model is shown in red. (c) Microscale thermophoresis (MST) binding data of spike with 683 fluorescently labelled Sb23, in the presence or absence of 200 nM ACE2. One representative 684 measurement is shown. Three independent measurements were performed and affinities of spike 685 to

Experimental SAXS data from Sb23 and its complex with RBD; (b) Distance distribution 705 functions of Sb23, RBD and their complex; (c) Two-phase MONSA shape of Sb23 (red beads) 706 and RBD (blue beads); (d) Hybrid model of

Locally-sharpened Coulomb potential map and cartoon model of Sb23 bound to the spike 724 protein in the '2-up' conformation. (b) Locally-sharpened Coulomb potential map and cartoon 725 model of Sb23 bound to the spike protein in the '1-up' conformation. (c) Cartoon model of Sb23-726 bound Spike in the '2-up' (left) '1-up' (right) conformation showing how ACE2 binding is blocked 727 by

 585 We thank the Sample Preparation and Characterization facility at EMBL (Hamburg, Germany) for 586 their support with nanoDSF, MST and biolayer interferometry measurements. We thank Stephan