A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

Abstract

SARS-CoV-2, the causative agent of COVID-19 ¹ , recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE2 ^2–7 . Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development ^8–18 . Llama-derived single-domain antibodies (nanobodies, ∼15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization ¹⁹ , which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity ( K _D = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC ₅₀ = 0.40 μg mL ⁻¹ ). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC ₅₀ = 12 ng mL ⁻¹ ) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

SciScore for 10.1101/2020.06.09.143438: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: Ethics Statement: The animal experiments were approved by the Institutional Animal Care and Use Committee of the Institut Pasteur of Shanghai, Chinese Academy of Sciences (Animal protocol No. A2020009) for in vivo stability assays, and by the Ethics Committees of Institute of Microbiology, Chinese Academy of Sciences (SQIMCAS2020010) for the live virus-related work. IRB: Ethics Statement: The animal experiments were approved by the Institutional Animal Care and Use Committee of the Institut Pasteur of Shanghai, Chinese Academy of Sciences (Animal protocol No. A2020009) for in vivo stability assays, and by the Ethics Committees of Institute of Microbiology, Chinese Academy of Sciences (SQIMCAS2020010) for the live virus-related work.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	In vivo stability of sybody in mice: The female 7-week-old ICR mice weighing 27 ± 1 g were intraperitoneally injected with phosphate buffered saline (PBS) or sybodies MR3, MR3-MR3, or MR3-MR3-ABD at 25mg kg−1 in a final volume of 100 μL in PBS.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The plate was washed three times using TBS before added with anti-myc antibodies at 1:2,000 dilution in TBS-BSA-T buffer (TBS supplemented with 0.5 %(w/v) BSA and 0.05 %(v/v	anti-myc suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Expi293 cells at density of 2.3 million per milliliter were transfected with the plasmid (final concentration of 2 mg L−1) using linear polyethylenimine (average MW of 25 kDa, 4 mg L−1)	Expi293 suggested: RRID:CVCL_D615)
Pseudotyped particle production and neutralizing assays: The retroviral pseudotyped particles were generated by co-transfection of HEK293T cells using polyethylenimine with the expression vectors encoding the various viral envelope glycoproteins, the Murine leukemia virus core/packaging components (MLV Gag-Pol), and a retroviral transfer vector harboring the gene encoding the green fluorescent protein (GFP).	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
VeroE6-hACE2 cells (104 cells/well) were seeded in a 48-well plate and infected 24 h later with 100 μL of virus supernatant in a final volume of 150 μL.	VeroE6-hACE2 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Mice challenge experiments: C57BL/6J female mice (6-8 weeks old) were treated with adenovirus serotype 5 expressing human angiotensin 1 converting enzyme 2 (hACE2) via the intranasal route as previously described40.	C57BL/6J suggested: None
Software and Algorithms
Sentences	Resources
The model was built with 2Fo-Fc maps in Coot 49, and refined using Phenix 50.	Coot suggested: (Coot, RRID:SCR_014222)
Structures were visualized using PyMol 51	PyMol suggested: (PyMOL, RRID:SCR_000305)

Results from OddPub: Thank you for sharing your data.

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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 5. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

Results from rtransparent:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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A potent synthetic nanobody targets RBD and protects mice from SARS-CoV-2 infection

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