Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies

  1. Maxwell A. Stefan
  2. Yooli K. Light
  3. Jennifer L. Schwedler
  4. Peter R. McIlroy
  5. Colleen M. Courtney
  6. Edwin A. Saada
  7. Christine E. Thatcher
  8. Ashlee M. Phillips
  9. Feliza A. Bourguet
  10. Catherine M. Mageeney
  11. Summer A. McCloy
  12. Nicole M. Collette
  13. Oscar A. Negrete
  14. Joseph S. Schoeniger
  15. Dina R. Weilhammer
  16. Brooke Harmon

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Abstract

The respiratory virus responsible for Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-2), has impacted nearly every aspect of life worldwide, claiming the lives of over 2.5 million people globally, at the time of this publication. Neutralizing nanobodies (V H H) represent a promising therapeutic intervention strategy to address the current SARS-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity V H H bacteriophage library, several potent neutralizing V H H antibodies were identified and evaluated for their capacity to tightly bind to the SARS-2 receptor-binding domain (RBD), to prevent binding of SARS-2 spike (S) to the cellular receptor Angiotensin-converting enzyme 2 (ACE2), and to neutralize viral infection. Preliminary preclinical evaluation of multiple nanobody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-2. The identified and characterized nanobodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic.

Author Summary

To fully address the on-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-2), it will be important to have both vaccines and therapeutic strategies to prevent and mitigate the effects of SARS-2. In this study, we describe the identification and characterization of potently neutralizing humanized single domain heavy chain (V H H) antibodies that have binding affinity for both the original Wuhan strain and widely circulating B.1.1.7/UK strain. V H H antibodies have the same therapeutic potential as conventional antibodies in half the size and with greater stability and solubility. Using a synthetic humanized high-diversity V H H phage library we identified several candidates with strong affinity for the SARS-2 spike that block the interaction of SARS-2 spike with the cellular receptor ACE2, and effectively neutralize infection with SARS-2 in vitro . By sequencing viral escape mutants generated in the presence of each V H H we mapped the binding sites of the V H H antibodies and assessed their affinity against newly emerging SARS-2 variants. Finally, we demonstrate that two of these V H H antibodies show prophylactic and therapeutic efficacy in vivo against challenge with SARS-2. This study establishes that screening highly diverse V H H phage libraries against viral threats can yield highly effective therapeutic agents in real time.

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  1. ScreenIT

    SciScore for 10.1101/2021.05.06.442911: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: (24) Animal Studies: All animal work was performed in accordance with protocols approved by the Lawrence Livermore National Laboratory Institutional Animal Care and Use Committee.
    Sex as a biological variableGroups of male and female K18-hACE2 C57BL/6J transgenic mice (Jackson Laboratory) ranging in age from 12-16 weeks were inoculated intranasally with 2.5×104 PFU SARS-2 (USA-WA 01/2020) while under anesthesia (4-5% isoflurane in 100% oxygen).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    ACE2-huFc, ACE2-rbFc, and VHH-huFc antibodies were produced by transient expression in CHO-S cells using the ExpiCHO expression system.
    VHH-huFc
    suggested: None
    A high diversity library was designed by incorporating the natural prevalence of amino acids at positions in CDR1 and CDR2 based off 670 functional VHH antibodies deposited on sdAb-DB (www.sdab-db.ca).(15) Amino acids cysteine and methionine were omitted from all CDR loops and full diversity was used for CDR3.
    CDR2
    suggested: None
    CDR3
    suggested: None
    Secondary antibody, goat anti-human H+L IgG HRP (Thermo) was added for an additional hour before washing.
    anti-human H+L IgG HRP
    suggested: None
    SARS-2-NG, VSV-SARS-2-GFP, or VSV-SARS-1-GFP was added to the serially diluted VHH-huFc antibodies for a final multiplicity of infection (MOI) of 0.2 (RG 2) or 0.1 (RG 3) and allowed to incubate at 37°C for 30 minutes to 1 hour with shaking prior to transfer of virus-VHH-huFc mixture to cells seeded in 96 (RG 3) or 384 well plates (RG 2).
    VSV-SARS-2-GFP
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    The Expi293 cells were subsequently infected with VSV-DG-GFP, itself pseudotyped with VSV-G, at 72 h post-transfection using a multiplicity of infection (MOI) of 3.
    Expi293
    suggested: RRID:CVCL_D615)
    VHH-huFc-virus complexes were incubated with Vero cells at 37°C with 5% CO2 for 12-16 h.
    Vero
    suggested: None
    After a 30-minute incubation of VHH-huFc-virus complexes with Vero E6 cells at 37°C with 5% CO2, overlays of 2 mL per well of 0.6% microcrystalline cellulose (MCC, Sigma 435244), in 8% FBS, 1% P/S complemented 2X MEM were added.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Groups of male and female K18-hACE2 C57BL/6J transgenic mice (Jackson Laboratory) ranging in age from 12-16 weeks were inoculated intranasally with 2.5×104 PFU SARS-2 (USA-WA 01/2020) while under anesthesia (4-5% isoflurane in 100% oxygen).
    C57BL/6J
    suggested: None
    Recombinant DNA
    SentencesResources
    DNA Manipulation and Production of Proteins: pSF-CMV-SARS-2-S was constructed as follows.
    pSF-CMV-SARS-2-S
    suggested: None
    pSF-SARS-2-RBD was produced by commercial production of double stranded DNA encoding a N-terminal Kozak, a signal peptide (MDWTWRFLFVVAAATGVQS), 319-577 of the SARS-2 S gene (GenBank:MN908947.3), a TEV site, and a C-terminal, decahistadine tag.
    pSF-SARS-2-RBD
    suggested: None
    All DNA fragments and pSF-CMV vector restriction digested with EcoRI and BamHI were assembled using NEBuilder HiFi DNA master.
    pSF-CMV
    suggested: None
    pAce2-huFc was produced by subcloning the ectodomain on human ACE2 (Sino Biological) into pCR-Fc using the NotI and BamHI restriction sites.
    pCR-Fc
    suggested: None
    To produce Ace2-rbFc (rabbit Fc domain), a gBlock (IDT) of this same region of Ace2 was subcloned into pFUSE rIgG-Fc2.
    pFUSE
    suggested: None
    The minimum region containing all 3 CDR domains, approximately 300 bps, was excised from the pADL20c backbone by two-step restriction digests, BglI followed by DdeI/BstEII double digests on the gel-purified small fragment from the BglI restriction reaction.
    pADL20c
    suggested: None
    Software and Algorithms
    SentencesResources
    Diversity of the nanobody library was determined by both NGS and colony PCR.
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    BCL files were converted to FASTQ and demutiplexed using the bcl2fastq script from MyIllumina (https://my.illumina.com).
    bcl2fastq
    suggested: (bcl2fastq , RRID:SCR_015058)
    The quality filtering and adaptor trimming were performed using fastp (https://github.com/OpenGene/fastp) with following parameters, -q 30 -l 100 -x 7.
    https://github.com/OpenGene/fastp
    suggested: (fastp, RRID:SCR_016962)
    R2read was reformatted to be reverse complemented and merged with R1 read using BBTools (BBMap, https://sourceforge.net/projects/bbmap/).(17) Three CDR domains with correct sequence lengths were extracted, concatenated and translated and counted unique amino acid sequences using a custom python script.
    BBMap
    suggested: (BBmap, RRID:SCR_016965)
    The dose response curves and 50% effective inhibitory concentrations (EC50) were generated using Graphpad Prism 9.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The filtered reads were mapped to Spike protein coding region of the SARS-2 Wuhan Hu-1 isolate (GenBank:MN908947.3) using Bowtie 2.
    Bowtie
    suggested: (Bowtie, RRID:SCR_005476)
    Kaplan-Meier survival curves were generated based on two independent experiments and log rank tests were performed with Bonferroni multiple comparison correction applied (GraphPad Prism).
    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: We detected the following sentences addressing limitations in the study:
    While vaccines have been great successes, therapeutic biologics are emerging as a critical tool in preventing progression to severe disease in those who do become infected.(31) Several therapeutic antibody candidates with efficacy against SARS-2 have been recently identified, including three with emergency use authorization, but there are a number of caveats associated with conventional antibodies as therapeutics such as time-consuming discovery, relying on immunized or patient sera, expensive and labor-intensive production, and the large doses required to achieve clinical efficacy. (9, 22, 32-34) High stability, solubility, and the ability to be multimerized, are just some of the many reasons why single-domain heavy chain-based antibody therapeutics (VHH-Fc) represent a highly promising method for treatment.(12) One VHH-based therapeutic is already approved by the FDA for treatment of a rare blood clotting disorder and many more are in late stage clinical trials.(12, 35) The small size of VHH antibodies and the wider distribution of CDR3 loop lengths compared to human IgGs expands the type of epitope which can be effectively targeted.(12) Finally, preliminary evidence suggests that VHH-Fc antibodies are transported more efficiently into the blood and lung parenchyma following intraperitoneal administration compared to conventional IgG1 antibodies.(36, 37) Several groups have recently used in vitro screening techniques to identify VHH domains with high affinity for the SARS-2...

    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: 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

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  2. Version published to 10.1101/2021.05.06.442911v1 on bioRxiv
  3. Version published to 10.1080/19420862.2021.1958663