An ultrapotent RBD-targeted biparatopic nanobody neutralizes broad SARS-CoV-2 variants

  1. Xiaojing Chi
  2. Xinhui Zhang
  3. Shengnan Pan
  4. Yanying Yu
  5. Yujin Shi
  6. Tianli Lin
  7. Huarui Duan
  8. Xiuying Liu
  9. Wenfang Chen
  10. Xuehua Yang
  11. Lan Chen
  12. Xiaoqian Dong
  13. Lili Ren
  14. Qiang Ding
  15. Jianwei Wang
  16. Wei Yang

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Abstract

The wide transmission and host adaptation of SARS-CoV-2 have led to the rapid accumulation of mutations, posing significant challenges to the effectiveness of vaccines and therapeutic antibodies. Although several neutralizing antibodies were authorized for emergency clinical use, convalescent patients derived natural antibodies are vulnerable to SARS-CoV-2 Spike mutation. Here, we describe the screen of a panel of SARS-CoV-2 receptor-binding domain (RBD) targeted nanobodies (Nbs) from a synthetic library and the design of a biparatopic Nb, named Nb1–Nb2, with tight affinity and super-wide neutralization breadth against multiple SARS-CoV-2 variants of concern. Deep-mutational scanning experiments identify the potential binding epitopes of the Nbs on the RBD and demonstrate that biparatopic Nb1–Nb2 has a strong escape-resistant feature against more than 60 tested RBD amino acid substitutions. Using pseudovirion-based and trans-complementation SARS-CoV-2 tools, we determine that the Nb1–Nb2 broadly neutralizes multiple SARS-CoV-2 variants at sub-nanomolar levels, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Lambda (C.37), Kappa (B.1.617.1), and Mu (B.1.621). Furthermore, a heavy-chain antibody is constructed by fusing the human IgG1 Fc to Nb1–Nb2 (designated as Nb1–Nb2-Fc) to improve its neutralization potency, yield, stability, and potential half-life extension. For the new Omicron variant (B.1.1.529) that harbors unprecedented multiple RBD mutations, Nb1–Nb2-Fc keeps a firm affinity (KD < 1.0 × 10 −12  M) and strong neutralizing activity (IC 50  = 1.46 nM for authentic Omicron virus). Together, we developed a tetravalent biparatopic human heavy-chain antibody with ultrapotent and broad-spectrum SARS-CoV-2 neutralization activity which highlights the potential clinical applications.

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  1. Version published to 10.1038/s41392-022-00912-4
  2. ScreenIT

    SciScore for 10.1101/2021.12.25.474052: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After 4 rounds of panning, phage ELISA identification was performed with 960 individual colonies using Anti-CM13 antibody in the plates coated with recombinant RBDs.
    Anti-CM13
    suggested: None
    Antibody-escape sites visualization: The RBD and ACE2 binding crystal structure (PDB: 6M0J) is represented by a surface pattern.
    ACE2
    suggested: None
    The antibody-escape amino acids on RBD are colored at each site.
    antibody-escape
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and reagents: The HEK293T (human kidney epithelial) cells were obtained from China Infrastructure of Cell Line Resource (Beijing, China).
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    The human hepatoma cell line Huh7 was obtained from Apath, Inc (Brooklyn, NY, USA) with permission from Dr. Charles Rice (Rockefeller University).
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Serially diluted antibodies were mixed with SARS-CoV-2 and inoculated into Caco-2-N cells.
    Caco-2-N
    suggested: None
    Recombinant DNA
    SentencesResources
    The plasmid DNA transfection mixture (1 ml) was composed of 15 μg of pNL-4.3-Luc-E-R- and 15 μg of pcDNA-SARS-CoV-2-S that was purchased from Sino Biologicals and reconstructed by deletion of 18 amino acid cytoplasmic tail.
    pcDNA-SARS-CoV-2-S
    suggested: None
    Substitutions of the residues at the sites selected for mutagenesis were based on the pcDNA3.1-SARS-CoV-2-S (GenBank: MN_908947) , which was purchased from Sino Biologicals and reconstructed by deletion of 18 amino acid cytoplasmic tail.
    pcDNA3.1-SARS-CoV-2-S
    suggested: None
    For the variants derived pseudovirus, the spike genes were codon optimized, synthesized, and cloned into pCAGGS vector.
    pCAGGS
    suggested: RRID:Addgene_127347)
    Software and Algorithms
    SentencesResources
    Statistics and reproducibility: Data were analyzed using GraphPad Prism 6.01 (GraphPad Software, San Diego, CA, USA).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.12.25.474052v1 on bioRxiv