Stable nebulization and muco‐trapping properties of regdanvimab/ IN ‐006 support its development as a potent, dose‐saving inhaled therapy for COVID ‐19

  1. Morgan D. McSweeney
  2. Ian Stewart
  3. Zach Richardson
  4. Hyunah Kang
  5. Yoona Park
  6. Cheolmin Kim
  7. Karthik Tiruthani
  8. Whitney Wolf
  9. Alison Schaefer
  10. Priya Kumar
  11. Harendra Aurora
  12. Jeff Hutchins
  13. Jong Moon Cho
  14. Anthony J. Hickey
  15. Soo Young Lee
  16. Samuel K. Lai

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Abstract

The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID‐19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self‐dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc‐mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID‐19 in several countries, can effectively trap SARS‐CoV‐2 virus‐like particles in fresh human airway mucus. IN‐006, a reformulation of regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN‐006 resulted in 100‐fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN‐006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS‐CoV‐2 and other respiratory pathologies.

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  1. Version published to 10.1002/btm2.10391
  2. ScreenIT

    SciScore for 10.1101/2022.02.27.482162: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Multiple particle tracking for quantifying mobility of SARS-CoV-2 virus like particles in AM treated with IN-006: Fresh, undiluted human AM were collected from extubated and otherwise would-be-discarded endotracheal tubes, as previously described [59], via a non-human subjects research designed protocol approved by the University of North Carolina at Chapel Hill.
    Sex as a biological variableRat nebulization study: To determine the concentrations of IN-006 achievable in vivo following inhaled dosing, we treated male and female Sprague Dawley Rats (age ~9-10 weeks at start of treatment) with nebulized IN-006 daily in an inhalation chamber, for 7 days.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The detection antibody was a peroxidase-conjugated goat anti-human IgG Fc antibody (Rockland 709-1317), used at a 1:5000 dilution in 1% milk in PBS, and was incubated at room temperature for 1hr.
    anti-human IgG
    suggested: (Rockland Cat# 709-1317, RRID:AB_217641)
    Experimental Models: Cell Lines
    SentencesResources
    Mixture of serially diluted antibodies at 3-fold ratio from 100 to 0.005 ng/ml and diluted pseudovirus to a predetermined number of copies was incubated for 1 hour and was added to stably expressing human ACE-2 HEK293T cells which are seeded into 96-well plate the day before the test.
    HEK293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Rat nebulization study: To determine the concentrations of IN-006 achievable in vivo following inhaled dosing, we treated male and female Sprague Dawley Rats (age ~9-10 weeks at start of treatment) with nebulized IN-006 daily in an inhalation chamber, for 7 days.
    Sprague Dawley
    suggested: None
    Recombinant DNA
    SentencesResources
    Production of fluorescent VLPs were made by co-transfection of pGAG-mCherry plasmid (kind gift from Gummuluru lab) and Cov2 S protein plasmid in a 1:1
    pGAG-mCherry
    suggested: None
    Non-replicating lentivirus pseudotyped with SARS-CoV-2 UK spike protein was created using the following plasmids, in a 1:1:1:2 ratio: pMDLg/pRRE, pRSV-REV, SARS-CoV-2 UK Spike, and pLL7 GFP.
    pMDLg/pRRE
    suggested: RRID:Addgene_12251)
    pRSV-REV
    suggested: RRID:Addgene_12253)
    pLL7
    suggested: RRID:Addgene_112133)
    Pseudovirus Neutralization: SARS-CoV-2 spike mutant pseudovirus was produced by transfection into HEK-293T with plasmid mixture such as third-generation Lentiviral packaging vectors pMDLg/pRRE, pRSV-Rev, pCDH-CMV-Nluc-copGFP-Puro, a dual reporter vector expressing luciferase and GFP, and the pCMV3-SARS-CoV-2 spike plasmid expressing the mutant SARS-CoV-2 spike prepared through each site-direct mutagenesis.
    HEK-293T
    suggested: None
    pCDH-CMV-Nluc-copGFP-Puro
    suggested: None
    pCMV3-SARS-CoV-2
    suggested: None
    Software and Algorithms
    SentencesResources
    Videos of VLPs diffusing in AM were recorded with MetaMorph software (Molecular Devices, Sunnyvale, CA) at a temporal resolution of 66.7 ms.
    MetaMorph
    suggested: None
    Finally, IC50 value was calculated using a nonlinear regression model by GraphPad Prism 5.0.
    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

    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.

  3. Version published to 10.1101/2022.02.27.482162v1 on bioRxiv