SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

  1. Federico Bertoglio
  2. Doris Meier
  3. Nora Langreder
  4. Stephan Steinke
  5. Ulfert Rand
  6. Luca Simonelli
  7. Philip Alexander Heine
  8. Rico Ballmann
  9. Kai-Thomas Schneider
  10. Kristian Daniel Ralph Roth
  11. Maximilian Ruschig
  12. Peggy Riese
  13. Kathrin Eschke
  14. Yeonsu Kim
  15. Dorina Schäckermann
  16. Mattia Pedotti
  17. Philipp Kuhn
  18. Susanne Zock-Emmenthal
  19. Johannes Wöhrle
  20. Normann Kilb
  21. Tobias Herz
  22. Marlies Becker
  23. Martina Grasshoff
  24. Esther Veronika Wenzel
  25. Giulio Russo
  26. Andrea Kröger
  27. Linda Brunotte
  28. Stephan Ludwig
  29. Viola Fühner
  30. Stefan Daniel Krämer
  31. Stefan Dübel
  32. Luca Varani
  33. Günter Roth
  34. Luka Čičin-Šain
  35. Maren Schubert
  36. Michael Hust

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Abstract

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

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  1. Version published to 10.1038/s41467-021-21609-2
  2. Version published to 10.1101/2020.06.05.135921v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.06.05.135921: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Antibodies
    SentencesResources
    Design of expression vectors: Production in Expi293F cells was performed using pCSE2.5-His-XP, pCSE2.6-hFc-XP or pCSE2.6-mFc-XP 68 where the respective single chain variable fragment of the antibodies or antigens were inserted by NcoI/NotI (NEB Biolabs) digestion.
    pCSE2.5-His-XP
    suggested: None
    pCSE2.6-hFc-XP
    suggested: None
    Production of antigens and scFv-Fc in mammalian cells: Antibodies, different domains or subunits of the Spike protein and the extracellular domain of ACE2 were produced in Expi293F cells (Thermo Fisher
    ACE2
    suggested: None
    All antigens, antibodies and scFv-Fc were run on Superdex 200 Increase 10/300GL (Cytiva) on Äkta or HPLC (Techlab) on an AdvanceBio SEC 300Å 2.7 μm, 7.8×300 mm (Agilent) for quality control.
    antigens ,
    suggested: None
    Fc-tagged ligand versions were labelled indirectly by using goat-anti-mFc-APC (Dianova) or mouse anti-hFcγ-APC (Biolegend) antibody. 100, 50 and 25 nM of antigen were incubated with 5×105 ACE2-expressing or non-transfected Expi293F cells (negative control) 50 min on ice.
    anti-hFcγ-APC
    suggested: None
    Antibody structures and computational docking studies: The antibody structures were modelled according to the canonical structure method using the RosettaAntibody program 74 as previously described 75 and docked to the experimental structure of SARS-CoV-2 S protein receptor binding domain (RBD, PDBid: 6M17) 6.
    SARS-CoV-2 S protein receptor binding domain
    suggested: None
    The antibody/RBD complexes were structurally clustered and then selected according to the scoring function (an estimate of energetically favourable solutions) and agreement with the peptide mapping data.
    antibody/RBD
    suggested: None
    The measurement was performed in 3 steps - 1st step blocking solution (50% PBS BSA 1/mg/mL, 50% Superblock (Thermo Scientific - Cat No: 37515)); 2nd step neutralizing SARS-CoV-2 antibody 8μg/mL; 3rd step goat anti human Alexa 546 antibody 5μg/mL (Invitrogen - Cat No: A-21089).
    anti human Alexa 546
    suggested: (Thermo Fisher Scientific Cat# A-21089, RRID:AB_2535745)
    Experimental Models: Cell Lines
    SentencesResources
    S1-HIS proteins from SARS-CoV-2 (expressed in HEK cells), SARS-CoV-1, MERS
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    SARS-CoV-2 neutralization in cell culture: VeroE6 cells (ATCC CRL-1586) were seeded at a density of 6*104/well onto cell culture 96-well plates (Nunc, Cat.#167008).
    VeroE6
    suggested: None
    , Baculovirus (Sino Biological 40592-V08B); S1(D614G), SARS-CoV-2, HEK293 (Sino Biological 40591-V08H3);; S1-S2, SARS, HEK293 (Acro Biosystems SPN-S52H5); S1, SARS, HEK293 (Acro Biosystems SIN-S52H5); S1-RBD, SARS, HEK293 (Acro Biosystems SPD-S52H6); S1-RBD, MERS, Baculovirus (Sino Biological 40071-V08B1); S1, MERS, HEK293 (Sino Biological 40069-V08H); N, MERS, Baculovirus (Sino Biological 40068-V08B); S1, HCoV-229E, HEK293 (Acro Biosystems SIN-V52H4); S1, HCoV-NL63, HEK293 (Acro Biosystems SIN-V52H3); S1, HCoV-HKU1, HEK293 (Sino Biological
    HCoV-229E
    suggested: None
    HCoV-NL63
    suggested: RRID:CVCL_RW88)
    HCoV-HKU1
    suggested: None
    Software and Algorithms
    SentencesResources
    Monoclonal binders were sequenced and analyzed using VBASE2 (www.vbase2.org) 72 and possible glycosylation positions in the CDRS were analyzed according to Lu et al 73
    VBASE2
    suggested: (VBASE2, RRID:SCR_007082)
    Docking was performed using the RosettaDock 3.12 software 76 as previously described 77.
    RosettaDock
    suggested: (RosettaDock, RRID:SCR_013393)
    Image data was quantified with the IncuCyte S3 GUI tools measuring the decrease of confluence concomitant with the cytopathic effect of the virus in relation to uninfected controls and controls without antibody and analyzed with GraphPad Prism 8.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    With two rounds of treatment per patient, this is a grave limitation, since one donor can only provide material for 1-2 patients 42,43. Human or humanized monoclonal antibodies are a powerful alternative to polyclonal antibodies derived from convalescent plasma. Following this approach, the humanized antibody Palivizumab was approved in 2009 for treatment and prevention of Respiratory Syncytial Virus (RSV) infections 46. Other antibodies against viral diseases successfully tested in clinical studies are mAb114 and REGN-EB3 against Ebola disease 24. Phage display derived antibodies are typically well established medications: twelve such antibodies are approved by EMA/FDA at the time of writing, a significant increase compared to the six such antibodies approved in 2016 47. In this work, we used phage display to isolate monoclonal human antibodies capable of neutralizing SARS-CoV-2 from a universal, naive antibody gene library that was generated from healthy donors before the emergence of the SARS-CoV-2 virus. This allowed selection of human antibodies against this virus without the necessity to obtain material from COVID-19 infected individuals. While most antibodies against SARS-CoV-2 were obtained from convalescent patients with few exceptions 20,21,48,49 our approach demonstrates that human antibodies with functional properties matching those of the antibodies isolated from convalescent patients can be generated without the necessity to wait for material from COVID-19 infec...

    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 pages 47, 48, 50, 51 and 52. 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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  4. Version published to 10.1101/2020.06.05.135921v1 on bioRxiv