key: cord-0687155-z3srgzb3
authors: Hanke, Leo; Perez, Laura Vidakovics; Sheward, Daniel J.; Das, Hrishikesh; Schulte, Tim; Moliner-Morro, Ainhoa; Corcoran, Martin; Achour, Adnane; Hedestam, Gunilla B. Karlsson; Hällberg, B. Martin; Murrell, Ben; McInerney, Gerald M.
title: An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction
date: 2020-07-08
journal: bioRxiv
DOI: 10.1101/2020.06.02.130161
sha: 79f38cc92fc10ecce06fd42dbf99dee3e0208ac4
doc_id: 687155
cord_uid: z3srgzb3

SARS-CoV-2 is the etiologic agent of COVID-19, currently causing a devastating pandemic for which pharmacological interventions are urgently needed. The virus enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. The nanobody binds with high affinity in the low nM range to the RBD, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations and that Ty1 sterically hinders RBD-ACE2 binding. This 12.8 kDa nanobody does not need an Fc domain to neutralize SARS-CoV-2, and can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.

generated a phage display library and performed two consecutive rounds of phage display, followed by an cryo-EM structure determination of the prefusion-stabilized spike ectodomain in complex with Ty1. The Since ACE2 can only be bound by an RBD in the 'up' conformation, the current cryo-EM reconstruction 151 clearly shows that ACE2 binding is sterically hindered from two sides (Fig. 4c) . Specifically, ACE2 152 binding is blocked both by the Ty1 nanobody bound to the RBD in the 'up' conformation and the 153 neighboring RBD in the 'down' conformation. Hence, ACE2 binding is sufficiently hindered with any 154 two of the available three binding RBD sites in the spike trimer. 155

The current coronavirus pandemic has drastic consequences for the world's population, and vaccines, 157 antibodies or antivirals are urgently needed. Neutralizing antibodies can block virus entry at an early step 158 of infection and potentially protect individuals that are at high risk of developing severe disease. We 159 report the identification and characterization of a SARS-CoV-2 RBD-specific single domain antibody 160 fragment (nanobody) termed Ty1 that potently neutralizes the virus. We identified Ty1 by binding assay 161 after two consecutive rounds of phage display, simultaneously monitoring sequence enrichment by NGS. 162

Although Ty1 exhibited the greatest fold-enrichment in the NGS analysis, multiple additional nanobodies 163 exhibited enrichment of varying extent across both rounds. As the correlation between phage display 164 enrichment and neutralization is likely imperfect, further analyses of our libraries will likely yield other potent SARS-CoV-2 neutralizing nanobodies. In addition to neutralization activity, we also show that Ty1 166 can be used as a detection reagent in flow cytometry and immunofluorescence demonstrating its 167 suitability as a research tool and for diagnostics. 168 domain is required for neutralization of SARS-CoV-2, precluding expression in bacterial culture. Naive 175 libraries of human single-domain antibodies (sdAbs) have also been screened to identify SARS-CoV-2 176 spike-specific nanobodies 21,22 , but they lack detailed structural information. Ty1 represents the first 177 single-domain antibody isolated from an animal specifically immunized with a SARS-CoV-2 protein. 178

179 Future work will aim to improve the potency and potential efficacy of Ty1 through various strategies. For 180 example, mutational scanning may yield potency improvements to Ty1. Also, since Ty1 already 181 neutralizes as a monomeric protein, the generation of homodimeric or trimeric fusion constructs is 182 expected to further increase its neutralization activity. Indeed, fusion of Ty1 to a human IgG1-Fc 183 dramatically improved the IC50 of this molecule, to approximately 5 ng/ml (Fig. S2 ). Additional strategies 184 will explore linker-based constructs that chain multiple copies of Ty1 together, which may provide 185 similar improvements in potency while retaining the possibility of being expressed in bacteria. Ty1 may 186 additionally be a useful component of a bi-specific or tri-specific antibody, which could combine epitope 187 specificities to increase the mutational barrier to viral escape. Based on our work, we hope that Ty1 will 188 be investigated as a candidate for antiviral therapy. The nanobodies were cloned for expression in the pHEN plasmid with a C-terminal Sortase recognition 219 site (LPETG) and a 6xHIS tag. This plasmid was used to transform BL21 cells for periplasmic 220 expression. Expression was induced with 1 mM IPTG at OD600 = 0.6; cells were grown overnight at 221 30°C. Nanobodies were retrieved from the periplasm by osmotic shock and purified by Ni-NTA affinity 222 purification and size-exclusion chromatography. 223

For mammalian expression, the sequence encoding the nanobody Ty1 was cloned upstream of an human 224

IgG1. This plasmid was used to transiently transfect FreeStyle 293F cells using the FreeStyle MAX 225 reagent. The Ty1-Fc fusion was purified from filtered supernatant on Protein G Sepharose followed by 226 size-exclusion chromatography. 227

Biotinylated and fluorescent probes were generated using Sortase A as described here 24 and here 25 . In 228 brief, nanobodies were site-specifically biotinylated on the C-terminus using Sortase A 5M. Nanobody at Alpaca immunization. Alpaca immunization and phage display was performed similarly as described 241

here 26 and here 27 . In brief, the adult male alpaca Tyson at PreClinics, Germany, was immunized 4 times in 242 a 60-day immunization schedule. SARS-CoV-2 S1-sheep-Fc (Native Antigen Company, SKU: 243 REC31806) was used for the first two immunization, and SARS-CoV-2 RBD produced in FreeStyle 293F 244 cells was used for the last two immunizations. For ITC, proteins were exchanged to 2xHBS-buffer (50 mM HEPES, 300 mM NaCl, pH 7.5) and isolated 323 as single peak populations by Superdex-200 HR10/300 size-exclusion chromatography. ITC 324 measurements were performed using an ITC200 calorimeter (GE Healthcare). The cell temperature was were loaded into the cell and syringe at concentrations of 4 μM and 75 μM, respectively. Data and 327 binding parameters were analyzed using the MicroCal PeakITC software (Malvern). The integrated heat 328 versus molar ratio plots of the Ty1:RBD interactions were obtained by subtracting the Ty1 dilution heat 329 uptake from the binding data. The NP-VHH1:RBD data were only baseline-corrected, since dilution 330 effects were not evident.

to glow-discharged CryoMatrix holey grids with amorphous alloy film (Zhenjiang Lehua Technology) in 336 a Vitrobot Mk IV (Thermo Fisher Scientific) at 4 degrees and 100% humidity (blot 10 s, blot force 3). 337

Cryo-EM data collection was performed with EPU 2.7 (Thermo Fisher Scientific) using a Krios G3i 338 transmission-electron microscope (Thermo Fisher Scientific) operated at 300 keV in the Karolinska 339

Institutet 3D-EM facility. Images were acquired in nanoprobe EFTEM mode with a slit width of 10 eV 340 using a GIF 967 energy filter (Ametek) and a K3 detector (Ametek) during 2.4 seconds with a dose rate of 

Cell entry mechanisms of SARS-CoV-2

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

Human neutralizing antibodies elicited by SARS-CoV-2 infection

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

Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput 379 single-cell sequencing of convalescent patients' B cells

Nanobodies and Nanobody-Based Human Heavy Chain 381

General strategy to humanize a camelid single-domain antibody and identification 383 of a universal humanized nanobody scaffold

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic

Determining kinetics and affinities of protein 402 interactions using a parallel real-time label-free biosensor, the Octet

Designing binding kinetic assay on the bio-layer interferometry (BLI)

biosensor to characterize antibody-antigen interactions

Characterization of Protein-Protein Interactions 407 by Isothermal Titration Calorimetry

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain 409

Structural characterisation of a nanobody derived from a naïve library that neutralises

Humanized Single Domain Antibodies Neutralize SARS-CoV-2 by Targeting Spike

Receptor Binding Domain

Identification of Human Single-Domain Antibodies against SARS-CoV-2

Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using 417

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation

Equilibrium analysis of high affinity interactions 461 using BIACORE

KI/SciLifeLabs) for providing access to BLI and ITC instruments. We also thank Fondation Dormeur

This work was supported by an EU grant 469 (CoroNAb) to BM, GM and GBKH and by project grants from the Swedish Research Council to BM

collected and processed cryo-EM data, built the model and performed the structural interpretation 475 of the data with the corresponding paper sections

conceived the study and wrote the initial draft

The authors declare no conflicts of interest. 480 481