Sybodies targeting the SARS-CoV-2 receptor-binding domain
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Abstract
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.
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SciScore for 10.1101/2020.04.16.045419: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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). α-mycsuggested: NoneExperimental Models: Cell Lines Sentences Resources This construct was expressed and secreted from Expi293 cells, and RBD-vYFP was extracted directly from culture medium supernatant using an immobilized … SciScore for 10.1101/2020.04.16.045419: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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). α-mycsuggested: NoneExperimental Models: Cell Lines Sentences Resources 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. Expi293suggested: RRID:CVCL_D615)A second purified RBD construct, consisting of SARS-CoV-2 residues Arg319—Phe541 fused to a murine IgG1 Fc domain (RBD-Fc) expressed in HEK293 cells, was purchased from Sino Biological (Catalogue number: 40592-V05H, 300 µg were ordered) HEK293suggested: NoneResults 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 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:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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