Novel ACE2-IgG1 fusions with improved in vitro and in vivo activity against SARS-CoV2

  1. Naoki Iwanaga
  2. Laura Cooper
  3. Lijun Rong
  4. Brandon Beddingfield
  5. Jackelyn Crabtree
  6. Ralph A. Tripp
  7. Xuebin Qin
  8. Jay K. Kolls

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Abstract

SARS-CoV2, the etiologic agent of COVID-19, uses ACE2 as a cell entry receptor. Soluble ACE2 has been shown to have neutralizing antiviral activity but has a short half-life and no active transport mechanism from the circulation into the alveolar spaces of the lung. To overcome this, we constructed an ACE2-human IgG1 fusion protein with mutations in the catalytic domain of ACE2. This fusion protein contained a LALA mutation that abrogates Fcrγ binding, but retains FcRN binding to prolong the half-life, as well as achieve therapeutic concentrations in the lung lavage. Interestingly, a mutation in the catalytic domain of ACE2, MDR504, completely abrogated catalytic activity, but significantly increased binding to SARS-CoV2 spike protein in vitro . This feature correlated with more potent viral neutralization in a plaque assay. Parental administration of the protein showed stable serum concentrations with a serum half-life of ∼ 145 hours with excellent bioavailability in the epithelial lining fluid of the lung. Prophylactic administration of MDR504 significantly attenuated SARS-CoV2 infection in a murine model. These data support that the MDR504 hACE2-Fc is an excellent candidate for pre or post-exposure prophylaxis or treatment of COVID-19.

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  1. Version published to 10.1101/2020.06.15.152157v2 on bioRxiv
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    SciScore for 10.1101/2020.06.15.152157: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All experiments were performed using sex- and age-matched controls and approved by the Institutional Animal Care and Use Committee of Tulane University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After incubation with IgG-HRP-conjugated anti-human antibody, membranes were washed and incubated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).
    anti-human
    suggested: None
    Pseudovirus Production: Pseudoviruses for antibody screening were generated using the following plasmids: S-Tag of SARS (pcDNA3.1(+)-SARS-S), S-Tag of SARS2 (pcDNA3.1(+)-SARS2-S), vesicular stomatitis virus (pcDNA3.1(+)-VSV-G), and the HIV-1 pro-viral vector pNL4-3.
    S-Tag of SARS (pcDNA3.1(+)-SARS-S), S-Tag of SARS2
    suggested: None
    We used purified anti-human IgG Fc antibody (Biolegend) as a capture antibody and anti-Human IgG Fc, Multi-Species SP ads-HRP (SouthrenBiotech) as a detection antibody and the other procedure is same as mentioned above.
    anti-human IgG
    suggested: None
    Tissues were blocked with 10% normal goat serum (NGS) for 40 minutes, followed by a 60 minute incubation with polyclonal guinea pig anti-SARS-CoV1 antibodies (1:1000) (NR-10361, BEI Resoruces).
    anti-SARS-CoV1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Coated plates were washed with washing buffer (0.05% Tween 20 in PBS), blocked for 2 h at room temperature with blocking buffer (1% BSA and 0.1% Tween 20 in PBS), and washed before the addition of the supernatants or cell lysates from transfected 293T cells.
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Human ACE2 neutralization of SARS-CoV2 by plaque assay: Vero E6 cells were plated in a 6 well plate at 8 × 105 cells per well and incubated overnight.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: Female wild type C57BL/6J mice 6-10-week-old were used for in vivo studies.
    C57BL/6J
    suggested: None
    For this study, we utilized the murine model of SARS-CoV2 were C57Bl/6 mice were first inoculated with adenovirus encoding human ACE2 (Vector Biolabs).
    C57Bl/6
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis: Statistical analysis was performed with GraphPad Prism 8.0.
    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 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

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  3. Version published to 10.1101/2020.06.15.152157v1 on bioRxiv