An Innovative antibody-based Plug-and-Play strategy for SARS-CoV-2

  1. Gururaj Shivange
  2. Debananda Gogoi
  3. Jogender Tushir-Singh

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Abstract

The novel and highly pathogenic coronavirus (SARS-CoV-2) remains a public health threat worldwide. SARS-CoV-2 enters human host lung cells via its spike protein binding to angiotensin-converting enzyme 2 (ACE2) in a process critical dependent on host protease-mediated fusion event. Thus, effective targeted therapies blocking the first step of viral fusion and cellular entry remains a critical unmet medical need to overcome disease pathology. Here we engineered and describe an antibody-based novel and targeted plug-and-play strategy, which directly competes with the proteolytic activation function of SAR-CoV-2 spike protein. The described strategy involves the engineering of furin substrate residues in IgG1 Fc-extended flexible region of spike targeting antibody. Our results with spike receptor-binding domain (RBD) targeting CR3022 antibody support blockade of the viral function using proof of concept ACE2 overexpressing cells. Our study reveals analytical, safe, and selective mechanistic insights for SARS-CoV-2 therapeutic design and is broadly applicable to the future coronaviridae family members (including mutant variants) exploiting the host protease system for cellular entry.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.11.434589: (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

    Antibodies
    SentencesResources
    Source of Antibody sequences: The sequence of Homo sapiens anti-SARS-CoV targeting immunoglobulin heavy chain and light chain are available at GenBank: DQ168569.1 (CR3022 VH) and GenBank: DQ168570.1 (CR3022 VL).
    anti-SARS-CoV targeting immunoglobulin heavy chain
    suggested: None
    Recombinant antibody and IgG4-Fc antigen expression: Free style CHO-S cells (Invitrogen, Reagents Table) were cultured and maintained according to supplier’s recommendations (Life technologies) biologics using free style CHO expression system (life technologies) and as previously described(1, 2).
    IgG4-Fc
    suggested: None
    The area under the curve was calculated for each peak and a relative percent monomer fraction was determined as described earlier (1) Binding studies by ELISA: Binding specificity and affinity of various described IgG1’s and FuG1 antibodies (including linkered FuG1, FuG1-Lin) s were determined by ELISA using the recombinant extracellular domain of corresponding receptor/target antigen.
    FuG1-Lin
    suggested: None
    Membranes were washed three times in TBST and then incubated with anti-rabbit or anti-mouse secondary antibodies (1/10,000 dilution, coupled to peroxidase) for 1 hr at room temperature.
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    In case of syncytia inhibition experiment, various control and FuG1 antibodies were added with indicated concentrations (see figures and figure legends for specific concentration) in different set of experiments.
    FuG1
    suggested: None
    Flow cytometry: The cell surface expression of ACE2, Spike, DR5, FOLR1 etc. was analyzed by flow cytometry in various experiments (see figure and figure legends) after treatment with various control and FuG1 antibodies.
    ACE2
    suggested: None
    DR5
    suggested: None
    Determination of Inhibitory concentration (IC50 Value): For inhibitory concentration determination, different concentrations (0-1000 nM) of antibodies (22-IgG, 22-FuGi and IgG1 control) were pre-incubated with 7 μl of SARS-CoV-2 (Luc) lenti-pseudovirus in a total volume of 10 μl for 30 minutes.
    22-IgG
    suggested: None
    22-FuGi
    suggested: None
    IgG1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, BHK21 cells were seeded at a density of ~2×106 cells per well into a 24-well cell culture plate in 500 μl of DMEM media.
    BHK21
    suggested: None
    To transduce cells with pseudovirions, ~2 x105 293T cells were seeded in a 96 well plate 20-24hrs prior to infection.
    293T
    suggested: None
    Briefly, 293T-ACE2 cells were grown in 96 well plate.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    After incubation, virus/antibody mixed was added onto ACE2-HEK293 cells.
    ACE2-HEK293
    suggested: None
    Software and Algorithms
    SentencesResources
    Transfected cultures were harvested after 10 days and filtered through 0.2-micron PES membrane filters (Milipore Express Plus).
    Milipore Express Plus
    suggested: None
    The antibody affinities (Kd) were calculated by non-linear regression analysis using GraphPad Prism software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Cells were washed and flow cytometry was performed using FACSCalibur.
    FACSCalibur
    suggested: None
    The data was analyzed by FCS Express (De Novo Software) and FlowJo.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    Results from OddPub: Thank you for sharing your data.

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

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  2. Version published to 10.1101/2021.03.11.434589 on bioRxiv