Engineering SARS-CoV-2 neutralizing antibodies for increased potency and reduced viral escape

  1. Fangzhu Zhao
  2. Celina Keating
  3. Gabriel Ozorowski
  4. Namir Shaabani
  5. Irene M. Francino-Urdaniz
  6. Shawn Barman
  7. Oliver Limbo
  8. Alison Burns
  9. Panpan Zhou
  10. Michael J. Ricciardi
  11. Jordan Woehl
  12. Quoc Tran
  13. Hannah L. Turner
  14. Linghang Peng
  15. Deli Huang
  16. David Nemazee
  17. Raiees Andrabi
  18. Devin Sok
  19. John R. Teijaro
  20. Timothy A. Whitehead
  21. Andrew B. Ward
  22. Dennis R. Burton
  23. Joseph G. Jardine

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Abstract

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.

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    SciScore for 10.1101/2022.01.06.475303: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: The Scripps Research Institutional Animal Care and Use Committee (IACUC) approved all experimental procedures involving all the animals in accordance with Protocol #20-0003.
    Sex as a biological variableMale 12–13-week-old hamsters were infused with antibodies intraperitoneally as described previously23.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To improve resolution of the antibody epitope and paratope, the best refinement from each dataset (Spike with 2 Fabs bound for both CC6.30 and CC6.33) was subjected to C3 symmetry expansion and focused classifications, using a spherical mask around the expected Fab/RBD region of a single protomer and Relion 3D classification without alignments.
    C3
    suggested: None
    After washing, yeast cells were stained with FITC-conjugated chicken anti-C-Myc antibody (Immunology Consultants Laboratory, CMYC-45F)
    anti-C-Myc
    suggested: None
    AF405-conjugated anti-V5 antibody (made in house), and streptavidin-APC (Invitrogen, SA1005) in 1:100 dilution for 20 min at 4 °C.
    anti-V5
    suggested: None
    SA1005
    suggested: None
    After washing, alkaline phosphatase-conjugated goat anti-human IgG Fcγ secondary antibody (Jackson ImmunoResearch, 109-055-008) was added in 1:1000 dilution and incubated for 1 h at RT.
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-055-008, RRID:AB_2337601)
    Experimental Models: Cell Lines
    SentencesResources
    Human HEK293T cells (ATCC) were used for pseudovirus production.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    FreeStyle HEK293 cells (ThermoFisher) were used for recombinant S protein production.
    HEK293
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Vero-E6 cells (ATCC) were used for live virus plaque assay.
    Vero-E6
    suggested: None
    After incubation for 10 min at RT, transfection mixture was added to Expi293 cells at a density of ∼ 3 million cells/mL.
    Expi293
    suggested: RRID:CVCL_D615)
    Yeast cells were firstly spun down and washed with PBSA (PBS + 1% BSA), then incubated with biotinylated SARS-CoV-2 RBD or S or HEK cell membrane protein at several non-depleting concentrations respectively for at least 30 min at 4°C.
    HEK
    suggested: None
    After that, 50 µL of Hela-hACE2 cells at 10,000 cells/well with 20 µg/mL of Dextran were added onto each well of the plates.
    Hela-hACE2
    suggested: None
    HEp2 epithelial cell polyreactive assay: Reactivity to human epithelial type 2 (HEp2) cells was determined by indirect immunofluorescence on HEp2 slides (Hemagen, 902360) according to manufacturer’s instructions.
    HEp2
    suggested: None
    Recombinant DNA
    SentencesResources
    The libraries were displayed on the surface of yeast as molecular Fab using the yeast display vector pYDSI containing the bidirectional Gal1-10 promoter.
    pYDSI
    suggested: None
    In brief, MLV gag/pol backbone (Addgene, 14887), MLV-CMV-Luciferase plasmid (Addgene, 170575), and SARS-CoV-2-d18 (Genbank MN908947) or SARS-CoV-1-d28 (Genbank AAP13567) or SARS-CoV-2 VOC spike plasmid were incubated with transfection reagent Lipofectamine 2000 (Thermo Fisher, 11668027) following manufacturer’s instructions for 20 min at RT.
    MLV-CMV-Luciferase
    suggested: None
    RBD library generation and identification of escape mutants: Yeast display plasmids pJS697 and pJS699 used for surface display of Wuhan-Hu-1 S RBD N343Q were previously described60.
    pJS697
    suggested: RRID:Addgene_168778)
    pJS699
    suggested: RRID:Addgene_168779)
    Software and Algorithms
    SentencesResources
    Syrian hamsters: Golden Syrian hamsters were provided by Charles River Laboratories (CRL:LVG(SYR)) and housed at the Scripps Research Institute.
    Charles River Laboratories
    suggested: (Charles River Laboratories, RRID:SCR_003792)
    Additionally, the CC6.33 dataset contained thousands of ligand-free HMP7 Spike particles which were also imported into cryoSPARC for final non-uniform refinement, with or without symmetry (Figure S2C and S2D).
    cryoSPARC
    suggested: (cryoSPARC, RRID:SCR_016501)
    Once a high map-to-model agreement was reaching, as measured by EMRinger57, and geometries were optimized, as judged by MolProbity58, the models were fit into the non-uniform refinement full trimer maps and combined with a Spike model refined into the ligand-free map (PDB 6vxx was used as the initial model and HPM7 mutations were added manually in Coot following iterative rounds of Rosetta relaxed refinement and Coot manual editing).
    Coot
    suggested: (Coot, RRID:SCR_014222)
    The Phenix software suite59 was used for structure validation, and for editing and preparation of PDB files for deposition.
    Phenix
    suggested: (Phenix, RRID:SCR_014224)
    Neutralization IC50 values were calculated using “One-Site Fit LogIC50” regression in GraphPad Prism 8.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    First, we plotted a standard curve of serially diluted virus (3000, 1000, 333, 111, 37, 12, 4, 1 PFU) versus RLU using four-parameter logistic regression (GraphPad Prism 8.0).
    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

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  2. Version published to 10.1101/2022.01.06.475303v1 on bioRxiv