Highly potent anti-SARS-CoV-2 multivalent DARPin therapeutic candidates

  1. Marcel Walser
  2. Sylvia Rothenberger
  3. Daniel L. Hurdiss
  4. Anja Schlegel
  5. Valérie Calabro
  6. Simon Fontaine
  7. Denis Villemagne
  8. Maria Paladino
  9. Tanja Hospodarsch
  10. Alexandra Neculcea
  11. Andreas Cornelius
  12. Patricia Schildknecht
  13. Mirela Matzner
  14. Martin Hänggi
  15. Marco Franchini
  16. Yvonne Kaufmann
  17. Doris Schaible
  18. Iris Schlegel
  19. Chloe Iss
  20. Thamar Looser
  21. Susanne Mangold
  22. Christel Herzog
  23. Dieter Schiegg
  24. Christian Reichen
  25. Filip Radom
  26. Andreas Bosshart
  27. Andreas Lehmann
  28. Micha A. Haeuptle
  29. Alexander Zürcher
  30. Toni Vagt
  31. Gabriel Sigrist
  32. Marcel Straumann
  33. Karl Proba
  34. Niina Veitonmäki
  35. Keith M. Dawson
  36. Christof Zitt
  37. Jennifer Mayor
  38. Sarah Ryter
  39. Heyrhyoung Lyoo
  40. Chunyan Wang
  41. Wentao Li
  42. Ieva Drulyte
  43. Wenjuan Du
  44. H. Kaspar Binz
  45. Leon de Waal
  46. Koert J. Stittelaar
  47. Sarah Taplin
  48. Seth Lewis
  49. Daniel Steiner
  50. Frank J.M. van Kuppeveld
  51. Olivier Engler
  52. Berend-Jan Bosch
  53. Michael T. Stumpp
  54. Patrick Amstutz

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Abstract

Globally accessible therapeutics against SARS-CoV-2 are urgently needed. Here, we report the generation of the first anti-SARS-CoV-2 DARPin molecules with therapeutic potential as well as rapid large-scale production capabilities. Highly potent multivalent DARPin molecules with low picomolar virus neutralization efficacies were generated by molecular linkage of three different monovalent DARPin molecules. These multivalent DARPin molecules target various domains of the SARS-CoV-2 spike protein, thereby limiting possible viral escape. Cryo-EM analysis of individual monovalent DARPin molecules provided structural explanations for the mode of action. Analysis of the protective efficacy of one multivalent DARPin molecule in a hamster SARS-CoV-2 infection model demonstrated a significant reduction of pathogenesis. Taken together, the multivalent DARPin molecules reported here, one of which has entered clinical studies, constitute promising therapeutics against the COVID-19 pandemic.

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  1. Version published to 10.1101/2020.08.25.256339v3 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.08.25.256339: (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
    The extract of each lysed clone was applied as a 1:200 dilution (final concentration) in PBSTB (PBS supplemented with 0.1% Tween 20® and 0.2% (w/v) BSA, pH 7.4) together with 20 nM (final concentration) biotinylated spike protein domain, 1:400 (final concentration) of anti-6His-D2 HTRF antibody – FRET acceptor conjugate (Cisbio) and 1:400 (final concentration) of anti-strep-Tb antibody FRET donor conjugate (Cisbio, France) to a well of a 384-well plate and incubated for 120 min at 4°C.
    biotinylated spike protein domain
    suggested: None
    anti-6His-D2 HTRF
    suggested: None
    anti-strep-Tb
    suggested: None
    Monoclonal antibodies against MERS-S (2), SARS-S or SARS2-S were included as a positive control(47).
    MERS-S ( 2
    suggested: None
    Serum concentrations were determined by sandwich ELISA using RBD as capture reagent and an anti-His-tag antibody as detection reagent and using a standard curve.
    anti-His-tag
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: Vero E6 cells (African green monkey kidney cells, ATCC® CRL1586™) purchased from ATCC (Manassas, VA 20110 USA) were passaged in cell culture medium DMEM (FG0445) containing 10% FBS and supplements (2mM L-Glutamine, Non-essential amino acids and 100 U/ml Penicillin 100 μg/ml Streptomycin and HEPES, all from Biochrom, Berlin, Germany) at 37°C with CO2.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Briefly, HEK-293T cells were transfected with pCAGGS expression vectors encoding MERS-S, SARS-S or SARS2-S carrying a 16-, 28- or 18-a.a. cytoplasmic tail truncation, respectively.
    HEK-293T
    suggested: None
    Twenty-four hours later, supernatants containing SARS2-S pseudotyped VSV particles were harvested and titrated on African green monkey kidney Vero E6 (ATCC#CRL-1586) cells.
    E6
    suggested: None
    Software and Algorithms
    SentencesResources
    The raw data (relative light unit values) were exported to GraphPad Prism v8.01, and the % neutralization data were normalized to the untreated PsV signal.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Image processing: Movie stacks were manually inspected and then imported in Relion version 3.0.1(48).
    Relion
    suggested: (RELION, RRID:SCR_016274)
    Drift and gain correction were performed with MotionCor2(49), and GCTF(50) was used to estimate the contrast transfer function for each movie.
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    , PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC) and BioRender (BioRender.com)
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    BioRender
    suggested: (Biorender, RRID:SCR_018361)
    (Certara, Princeton, USA) or GraphPadPrism (GraphPad Software, La Jolla,USA) and non-compartmental analyses.
    GraphPad
    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:
    A number of alternative molecules are being developed to complement and partially overcome this limitation of antibodies. Here we describe the generation of DARPin molecules - one prominent alternative to antibodies(30) amongst others(31–34) - that bind the SARS-CoV-2 spike protein. We tested a library of one trillion DARPin molecules and identified multiple DARPin molecules with different functionalities and binding specificities. By molecular linkage of individual DARPin molecules, we developed multi-DARPin molecules with low picomolar neutralizing activity and demonstrated their protective efficacy against SARS-CoV-2 infection in a hamster model. In particular, reduced lung tissue damage and reduced virus replication in the lower and upper respiratory tract were observed, the latter also being important for reducing virus shedding and transmission. The most advanced of those multi-DARPin molecules, MP0420 or ensovibep, is currently being studied in Phase 1. The most potent neutralizing mono-DARPin molecules were found to target the RBD, blocking the spike-ACE2 interaction necessary for infection. This finding is congruent with the identified epitopes of potent neutralizing antibodies obtained from COVID-19 patients(20, 35-39). Thus, the in vitro approach using DARPin molecules independently confirms that the ACE2 interaction site on the SARS-CoV-2 spike protein is one of the most vulnerable sites to block virus infection in cell culture. The single-chain binding domain nat...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 14 and 30. 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.

    About SciScore

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  3. Version published to 10.1101/2020.08.25.256339v2 on bioRxiv
  4. Version published to 10.1101/2020.08.25.256339v1 on bioRxiv