A high-affinity RBD-targeting nanobody improves fusion partner’s potency against SARS-CoV-2

  1. Hebang Yao
  2. Hongmin Cai
  3. Tingting Li
  4. Bingjie Zhou
  5. Wenming Qin
  6. Dimitri Lavillette
  7. Dianfan Li

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Abstract

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

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  1. Version published to 10.1371/journal.ppat.1009328
  2. ScreenIT

    SciScore for 10.1101/2020.09.24.312595: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Pseudotyped particle production and neutralizing assay: To generate retroviral pseudotyped particles, HEK293T cells were co-transfected with the vectors expressing the various viral envelope glycoproteins, the murine leukemia virus core/packaging components (MLV Gag-Pol), and a retroviral transfect vector expressing the green fluorescence protein (GFP).
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Fifty microliters of VeroE6-hACE2 cells (104 cells/well) were seeded in a 48-well plate.
    VeroE6-hACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    The models were manually adjusted as guided by the 2Fo-Fc maps in Coot (48), and refined using Phenix (49).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    Structures were visualized using PyMol (50).
    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 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.
    • 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.

    About SciScore

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  3. Version published to 10.1101/2020.09.24.312595v1 on bioRxiv