An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

  1. Michael Schoof
  2. Bryan Faust
  3. Reuben A. Saunders
  4. Smriti Sangwan
  5. Veronica Rezelj
  6. Nick Hoppe
  7. Morgane Boone
  8. Christian B. Billesbølle
  9. Cristina Puchades
  10. Caleigh M. Azumaya
  11. Huong T. Kratochvil
  12. Marcell Zimanyi
  13. Ishan Deshpande
  14. Jiahao Liang
  15. Sasha Dickinson
  16. Henry C. Nguyen
  17. Cynthia M. Chio
  18. Gregory E. Merz
  19. Michael C. Thompson
  20. Devan Diwanji
  21. Kaitlin Schaefer
  22. Aditya A. Anand
  23. Niv Dobzinski
  24. Beth Shoshana Zha
  25. Camille R. Simoneau
  26. Kristoffer Leon
  27. Kris M. White
  28. Un Seng Chio
  29. Meghna Gupta
  30. Mingliang Jin
  31. Fei Li
  32. Yanxin Liu
  33. Kaihua Zhang
  34. David Bulkley
  35. Ming Sun
  36. Amber M. Smith
  37. Alexandrea N. Rizo
  38. Frank Moss
  39. Axel F. Brilot
  40. Sergei Pourmal
  41. Raphael Trenker
  42. Thomas Pospiech
  43. Sayan Gupta
  44. Benjamin Barsi-Rhyne
  45. Vladislav Belyy
  46. Andrew W. Barile-Hill
  47. Silke Nock
  48. Yuwei Liu
  49. Nevan J. Krogan
  50. Corie Y. Ralston
  51. Danielle L. Swaney
  52. Adolfo García-Sastre
  53. Melanie Ott
  54. Marco Vignuzzi
  55. QCRG Structural Biology Consortium
  56. Peter Walter
  57. Aashish Manglik
  58. Caleigh M. Azumaya
  59. Cristina Puchades
  60. Ming Sun
  61. Julian R. Braxton
  62. Axel F. Brilot
  63. Meghna Gupta
  64. Fei Li
  65. Kyle E. Lopez
  66. Arthur Melo
  67. Gregory E. Merz
  68. Frank Moss
  69. Joana Paulino
  70. Thomas H. Pospiech
  71. Sergei Pourmal
  72. Alexandrea N. Rizo
  73. Amber M. Smith
  74. Paul V. Thomas
  75. Feng Wang
  76. Zanlin Yu
  77. Miles Sasha Dickinson
  78. Henry C. Nguyen
  79. Daniel Asarnow
  80. Melody G. Campbell
  81. Cynthia M. Chio
  82. Un Seng Chio
  83. Devan Diwanji
  84. Bryan Faust
  85. Meghna Gupta
  86. Nick Hoppe
  87. Mingliang Jin
  88. Junrui Li
  89. Yanxin Liu
  90. Gregory E. Merz
  91. Smriti Sangwan
  92. Tsz Kin Martin Tsui
  93. Raphael Trenker
  94. Donovan Trinidad
  95. Eric Tse
  96. Kaihua Zhang
  97. Fengbo Zhou
  98. Nadia Herrera
  99. Huong T. Kratochvil
  100. Ursula Schulze-Gahmen
  101. Michael C. Thompson
  102. Iris D. Young
  103. Justin Biel
  104. Ishan Deshpande
  105. Xi Liu
  106. Christian Bache Billesbølle
  107. Carlos Nowotny
  108. Amber M. Smith
  109. Jianhua Zhao
  110. Alisa Bowen
  111. Nick Hoppe
  112. Yen-Li Li
  113. Phuong Nguyen
  114. Mali Safari
  115. Kaitlin Schaefer
  116. Natalie Whitis
  117. Michelle Moritz
  118. Tristan W. Owens
  119. Amy Diallo
  120. Kate Kim
  121. Jessica K. Peters
  122. Erron W. Titus
  123. Jenny Chen
  124. Loan Doan
  125. Sebastian Flores
  126. Victor L. Lam
  127. Yang Li
  128. Megan Lo
  129. Aye C. Thwin
  130. Stephanie Wankowicz
  131. Yang Zhang
  132. David Bulkley
  133. Arceli Joves
  134. Almarie Joves
  135. Liam McKay
  136. Mariano Tabios
  137. Oren S. Rosenberg
  138. Kliment A. Verba
  139. David A. Agard
  140. Yifan Cheng
  141. James S. Fraser
  142. Adam Frost
  143. Natalia Jura
  144. Tanja Kortemme
  145. Nevan J. Krogan
  146. Aashish Manglik
  147. Daniel R. Southworth
  148. Robert M. Stroud

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Abstract

Monoclonal antibodies that bind to the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) show therapeutic promise but must be produced in mammalian cells and need to be delivered intravenously. By contrast, single-domain antibodies called nanobodies can be produced in bacteria or yeast, and their stability may enable aerosol delivery. Two papers now report nanobodies that bind tightly to spike and efficiently neutralize SARS-CoV-2 in cells. Schoof et al. screened a yeast surface display of synthetic nanobodies and Xiang et al. screened anti-spike nanobodies produced by a llama. Both groups identified highly potent nanobodies that lock the spike protein in an inactive conformation. Multivalent constructs of selected nanobodies achieved even more potent neutralization.

Science , this issue p. 1473 , p. 1479

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  1. ScreenIT

    SciScore for 10.1101/2020.08.08.238469: (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.

    Table 2: Resources

    No key resources detected.

    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.

    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.

  2. Version published to 10.1126/science.abe3255
  3. Version published to 10.1101/2020.08.08.238469v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.08.08.238469: (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
    B, Nanobody inhibition of 1 nM Spike*Alexa 647 binding to ACE2 expressing HEK293T cells after incubation at either 25 °C or 50 °C for 1 hour or after aerosolization.
    HEK293T
    suggested: None
    We used a previously described construct to express and purify the pre-fusion SARS-CoV-2 Spike ectodomain (Spike*) [15]. ExpiCHO or Expi293T cells (ThermoFisher) were transfected with the Spike* construct per the manufacturer’s instructions for the MaxTiter protocol and harvested between 3-9 days after transfection.
    Expi293T
    suggested: None
    The mixture was then used to inoculate Vero E6 cells seeded in 12-well plates, for one hour at 37 °C, 5% CO2.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    kanamycin pPW3546 Nb6 pet-26b(+) kanamycin pPW3547 Nb8 pet-26b(+) kanamycin pPW3548 Nb11 pet-26b(+) kanamycin pPW3549 Nb12 pet-26b(+) kanamycin pPW3550 Nb15 pet-26b(+) kanamycin pPW3551 Nb16 pet-26b(+) kanamycin pPW3552 Nb17 pet-26b(+) kanamycin pPW3553 Nb18 pet-26b(+) kanamycin pPW3554 Nb19 pet-26b(+) kanamycin pPW3555 Nb24 pet-26b(+) kanamycin pPW3557 Trivalent Nb6, 20AA length GS linker pet-26b(+) kanamycin pPW3558 Trivalent Nb3, 15AA length GS linker pet-26b(+) kanamycin pPW3559 Trivalent Nb11, 15AA length GS linker pet-26b(+) kanamycin pPW3560 Bivalent Nb3, 15AA length GS linker pet-26b(+) kanamycin pPW3561 Bivalent Nb6, 15AA length GS linker pet-26b(+) kanamycin pPW3563 Trivalent mNb6, 20AA length GS linker pet-26b(+) kanamycin pPW3564 mNb6 pet-26b(+) kanamycin METHODS Expression and purification of SARS-CoV-2 Spike, RBD, and ACE2.
    Nb11 pet-26b(+ ) kanamycin pPW3549 Nb12 pet-26b(+ ) kanamycin pPW3550 Nb15 pet-26b(+ ) kanamycin pPW3551 Nb16 pet-26b(+ ) kanamycin pPW3552 Nb17 pet-26b(+ ) kanamycin pPW3553 Nb18 pet-26b(+ ) kanamycin pPW3554 Nb19 pet-26b(+ ) kanamycin pPW3555 Nb24 pet-26b(+
    suggested: None
    Software and Algorithms
    SentencesResources
    Extracted particles in 2D classes suggestive of various Spike* views were subject to a round of heterogenous refinement in cryoSPARC with two naïve classes generated from a truncated Ab initio job, and a 20 Å low-pass filtered volume of apo-Spike* in the closed conformation.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Particles in the Spike* 3D class were subject to 25 iterations of 3D classification into 6 classes without alignment in RELION, using the same input volume from cryoSPARC 3D classification, low pass filtered to 60 Å, T = 8.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Protein Ligand R.m.s. deviations Bond lengths (Å) Bond angles (°) Validation MolProbity score Clashscore Poor rotamers (%) EMRinger score CaBLAM score Ramachandran plot Favored (%) Allowed (%) Disallowed (%) Spike*-Nb6 Open Closed XXXX XXXX XXXX
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    Closed XXXX XXXX Titan Krios/Gatan K3 with Gatan Bioquantum Energy Filter SerialEM, 3x3 image shift 105,000 300 66 8 0.55 -0.8 to -2.0 0.834 (physical) 5,317 4,103 1,609 2,033,067 1,204,855 585,250 40,125 (cisTEM) C1 58,493 (cisTEM)
    cisTEM
    suggested: (cisTEM, RRID:SCR_016502)
    Each collection was performed with semi-automated scripts in SerialEM [46].
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Image Processing For all datasets, dose fractionated super-resolution movies were motion corrected with MotionCor2 [47].
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    Final models were analyzed in PHENIX, with statistics reported in Supplementary Table 1.
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    Lastly, the area under the curve MS1 intensities reported from FragPipe were summarized for each peptide species using MSstats [60].
    MSstats
    suggested: (MSstats, RRID:SCR_014353)
    Model building was performed with COOT and refined with PHENIX and BUSTER[54, 56, 63].
    COOT
    suggested: (Coot, RRID:SCR_014222)
    The number of plaques in quadruplicate wells for each dilution was used to determine the half maximal inhibitory concentrations (IC50) using 3-parameter logistic regression (GraphPad Prism version 8).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The resulting molar ellipticity values were normalized and plotted in GraphPad Prism 8.0 after applying a nearest neighbor smoothing function.
    GraphPad Prism
    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 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.

    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.

  5. ScreenIT

    SciScore for 10.1101/2020.08.08.238469: (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
    B, Nanobody inhibition of 1 nM Spike*Alexa 647 binding to ACE2 expressing HEK293T cells after incubation at either 25 °C or 50 °C for 1 hour or after aerosolization.
    HEK293T
    suggested: None
    Expi293T cells (ThermoFisher) were transfected with the RBD construct per the manufacturer’s instructions and harvested between 3-6 days after transfection.
    Expi293T
    suggested: None
    Neutralization of infectious SARS-CoV-2 was performed using a plaque reduction neutralization test in Vero E6 cells (CRL-1586, ATCC).
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    kanamycin pPW3546 Nb6 pet-26b(+) kanamycin pPW3547 Nb8 pet-26b(+) kanamycin pPW3548 Nb11 pet-26b(+) kanamycin pPW3549 Nb12 pet-26b(+) kanamycin pPW3550 Nb15 pet-26b(+) kanamycin pPW3551 Nb16 pet-26b(+) kanamycin pPW3552 Nb17 pet-26b(+) kanamycin pPW3553 Nb18 pet-26b(+) kanamycin pPW3554 Nb19 pet-26b(+) kanamycin pPW3555 Nb24 pet-26b(+) kanamycin pPW3557 Trivalent Nb6, 20AA length GS linker pet-26b(+) kanamycin pPW3558 Trivalent Nb3, 15AA length GS linker pet-26b(+) kanamycin pPW3559 Trivalent Nb11, 15AA length GS linker pet-26b(+) kanamycin pPW3560 Bivalent Nb3, 15AA length GS linker pet-26b(+) kanamycin pPW3561 Bivalent Nb6, 15AA length GS linker pet-26b(+) kanamycin pPW3563 Trivalent mNb6, 20AA length GS linker pet-26b(+) kanamycin pPW3564 mNb6 pet-26b(+) kanamycin METHODS Expression and purification of SARS-CoV-2 Spike, RBD, and ACE2.
    Nb11 pet-26b(+ ) kanamycin pPW3549 Nb12 pet-26b(+ ) kanamycin pPW3550 Nb15 pet-26b(+ ) kanamycin pPW3551 Nb16 pet-26b(+ ) kanamycin pPW3552 Nb17 pet-26b(+ ) kanamycin pPW3553 Nb18 pet-26b(+ ) kanamycin pPW3554 Nb19 pet-26b(+ ) kanamycin pPW3555 Nb24 pet-26b(+
    suggested: None
    Software and Algorithms
    SentencesResources
    Particles in the Spike* 3D class were subject to 25 iterations of 3D classification into 6 classes without alignment in RELION, using the same input volume from cryoSPARC 3D classification, low pass filtered to 60 Å, T = 8.
    RELION
    suggested: (RELION, RRID:SCR_016274)
    Particles in Spike* 2D classes were selected for a round of heterogeneous refinement in cryoSPARC using a 20 Å low-pass filtered volume of apo-Spike* in the closed conformation and additional naïve classes for removal of non-Spike* particles.
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Protein Ligand R.m.s. deviations Bond lengths (Å) Bond angles (°) Validation MolProbity score Clashscore Poor rotamers (%) EMRinger score CaBLAM score Ramachandran plot Favored (%) Allowed (%) Disallowed (%) Spike*-Nb6 Open Closed XXXX XXXX XXXX
    MolProbity
    suggested: (MolProbity, RRID:SCR_014226)
    Closed XXXX XXXX Titan Krios/Gatan K3 with Gatan Bioquantum Energy Filter SerialEM, 3x3 image shift 105,000 300 66 8 0.55 -0.8 to -2.0 0.834 (physical) 5,317 4,103 1,609 2,033,067 1,204,855 585,250 40,125 (cisTEM) C1 58,493 (cisTEM)
    cisTEM
    suggested: (cisTEM, RRID:SCR_016502)
    Each collection was performed with semi-automated scripts in SerialEM [46].
    SerialEM
    suggested: (SerialEM, RRID:SCR_017293)
    Image Processing For all datasets, dose fractionated super-resolution movies were motion corrected with MotionCor2 [47].
    MotionCor2
    suggested: (MotionCor2, RRID:SCR_016499)
    The final structure was inspected and manually adjusted in COOT and ISOLDE, followed by real space refinement in PHENIX [54-56].
    PHENIX
    suggested: (Phenix, RRID:SCR_014224)
    Lastly, the area under the curve MS1 intensities reported from FragPipe were summarized for each peptide species using MSstats [60].
    MSstats
    suggested: (MSstats, RRID:SCR_014353)
    Model building was performed with COOT and refined with PHENIX and BUSTER[54, 56, 63].
    COOT
    suggested: (Coot, RRID:SCR_014222)
    The number of plaques in quadruplicate wells for each dilution was used to determine the half maximal inhibitory concentrations (IC50) using 3-parameter logistic regression (GraphPad Prism version 8).
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    The resulting molar ellipticity values were normalized and plotted in GraphPad Prism 8.0 after applying a nearest neighbor smoothing function.
    GraphPad Prism
    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 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.

    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.

  6. Version published to 10.1101/2020.08.08.238469v1 on bioRxiv