Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

Abstract

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 IC ₅₀ 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 an unusual 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.

SciScore for 10.1101/2020.06.23.165415: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
Periplasmic extracts were transferred to a microtiter plates, previously coated with Protein A (Sigma), blocked in TBS-BSA buffer and subsequently incubated with anti-myc antibody (Sigma).	anti-myc suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Suspension adapted HEK293-F cells were grown in 600-ml Bioreactors in 250 ml of serum free FreeStyle medium in an incubator at 37°C with humidified atmosphere at 8% CO2 on an orbital shaker platform rotating at 220 rpm.	HEK293-F suggested: RRID:CVCL_6642)
Neutralization Assay: Pseudotyped neutralization assays were adapted from protocols previously …

SciScore for 10.1101/2020.06.23.165415: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
Periplasmic extracts were transferred to a microtiter plates, previously coated with Protein A (Sigma), blocked in TBS-BSA buffer and subsequently incubated with anti-myc antibody (Sigma).	anti-myc suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Suspension adapted HEK293-F cells were grown in 600-ml Bioreactors in 250 ml of serum free FreeStyle medium in an incubator at 37°C with humidified atmosphere at 8% CO2 on an orbital shaker platform rotating at 220 rpm.	HEK293-F suggested: RRID:CVCL_6642)
Neutralization Assay: Pseudotyped neutralization assays were adapted from protocols previously validated to characterize the neutralization of HIV 38, but with the use of HEK293T-ACE2 cells as previously described 25.	HEK293T-ACE2 suggested: None
Briefly, pseudotyped lentiviruses displaying the SARS-CoV-2 spike protein (harboring an 18 amino acid truncation of the cytoplasmic tail 39) and packaging a luciferase reporter gene were generated by the co-transfection of HEK293T cells.	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
Experimental Models: Organisms/Strains
Sentences	Resources
The gene for RBD expression comprises residues (319 − 566) of 2019-nCoV RBD-SD1 and was cloned into a pαH mammalian expression vector containing a secretion signal, a C-terminal Sortase recognition motif and a non-cleavable Histidine tag 25	RBD-SD1 suggested: None
Software and Algorithms
Sentences	Resources
Biophysical methods: Small angle X-ray scattering measurements (SAXS): The SAXS data were collected at the EMBL P12 beamline of the storage ring PETRA III (DESY, Hamburg, Germany) using a robotic sample changer 41.	EMBL suggested: (ChEMBL, RRID:SCR_014042)
The radial average was performed and solvent scattering was subtracted using the SASFLOW pipeline 42.	SASFLOW suggested: None
The distance distributions were computed by GNOM 43 and the overall parameters were determined from the reduced data using relevant programs from the ATSAS suite 42.	ATSAS suggested: (ATSAS, RRID:SCR_015648)
Hybrid models of the sybodies and of the RBD were constructed by CORAL 45 using the available high-resolution portions as rigid bodies and amending them by chains of dummy residues to represent the fragments not present in the crystal.	CORAL suggested: (Coral, RRID:SCR_011849)
Extracted particles were imported into cryoSPARC v2.15.0 49 for 2D classification, heterogenous refinement and non-uniform 3D refinement 50.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
The model was extended and manually adjusted in COOT 51.	COOT suggested: (Coot, RRID:SCR_014222)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2

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