Converting non-neutralizing SARS-CoV-2 antibodies targeting conserved epitopes into broad-spectrum inhibitors through receptor blockade

  1. Payton A.-B. Weidenbacher
  2. Eric Waltari
  3. Izumi de los Rios Kobara
  4. Benjamin N. Bell
  5. John E. Pak
  6. Peter S. Kim

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Abstract

All but one of the authorized monoclonal antibody-based treatments for SARS-CoV-2 are largely ineffective against Omicron, highlighting the critical need for biologics capable of overcoming SARS-CoV-2 evolution. These mostly ineffective therapeutic antibodies target epitopes that are not highly conserved. Here we describe broad-spectrum SARS-CoV-2 inhibitors developed by tethering the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2), to antibodies that are known to be non-neutralizing, but which target highly conserved epitopes in the viral spike protein. These inhibitors, called Re ceptor-blocking co nserved n on- n eutralizing A nti b o d ies (ReconnAbs), potently neutralize all SARS-CoV-2 variants of concern (VOC), including Omicron. Neutralization potency is dependent on both the binding and inhibitory ReconnAb components as activity is lost when the linker joining the two is severed. In addition, a bifunctional ReconnAb, made by linking ACE2 to a bispecific antibody targeting two non-overlapping conserved epitopes, defined here, shows sub-nanomolar neutralizing activity against all VOCs, including Omicron. Given their conserved targets and modular nature, ReconnAbs have the potential to act as broad- spectrum therapeutics against SARS-CoV-2 and other emerging pandemic diseases.

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  1. ScreenIT

    SciScore for 10.1101/2022.01.24.477625: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Library Design: A library of antibodies directed against SARS-CoV-2 Spike (S) protein was developed using paired antibody sequences, meaning antibody sequences for which the heavy and light chain are both known, from the Coronavirus Antibody Database, CoV-AbDab57.
    CoV-AbDab57
    suggested: None
    CrossMAb, antibody, hFc-ACE2, and Lentiviral plasmids were maxi prepped in the same fashion.
    hFc-ACE2
    suggested: None
    Two microliters of mouse anti-hexa His antibody (BioLegend) were added to the 10 mL sample and incubated for 1 h at room temperature.
    anti-hexa
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Viral transfections were done in HEK293T cells using calcium phosphate transfection reagent.
    HEK293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    The resultant 10mL was added to plated HEK cells from which the medium had been removed.
    HEK
    suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)
    Neutralization: The target cells used for infection in viral neutralization assays were from a HeLa cell line stably overexpressing the SARS-CoV-2 receptor, ACE2, as well as the protease known to process SARS-CoV-2, TMPRSS2.
    HeLa
    suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
    ACE2/TMPRSS2/HeLa cells were plated one day prior to infection at 5,000 cells per well.
    ACE2/TMPRSS2/HeLa
    suggested: None
    Recombinant DNA
    SentencesResources
    Equimolar aliquots of each scFv plasmid were pooled and the resultant pool was amplified using primers which annealed to the hexa-his Tag (reverse primer) or signal peptide (forward primer) and had a 50bp overlap with the pPNL6 vector digested with NheI and BamHI.
    scFv
    suggested: None
    Yeast were resuspended in electroporation buffer (10 mM Tris Base, 250 mM sucrose, 2mM MgCl2) containing the gel extracted library amplification and digested pPNL6 vector.
    pPNL6
    suggested: None
    Light chain (LC) and LC-ACE2 fusion proteins: Antibody sequences were cloned into the CMV/R plasmid backbone for expression under a CMV promoter.
    CMV/R
    suggested: None
    hCoV spike proteins constructs: Spike proteins from six hCoVs were cloned into a pADD2 vector between the rBeta-globin intron and β-globin poly(A).
    pADD2
    suggested: None
    Lentivirus plasmids: Plasmids encoding the full-length spike proteins with native signal peptides were cloned into the background of the HDM-SARS2-Spike-delta21 plasmid (Addgene Plasmid #155130).
    HDM-SARS2-Spike-delta21
    suggested: RRID:Addgene_155130)
    They are: pHAGE-Luc2- IRS-ZsGreen (NR-52516), HDM-Hgpm2 (NR-52517), pRC-CMV-Rev1b (NR-52519), and HDM- tat1b (NR-52518).
    pHAGE-Luc2- IRS-ZsGreen
    suggested: None
    The EtOH solution was then added to a geneJET plasmid miniprep column and the regular wash steps and elution steps were followed.
    geneJET
    suggested: None
    Digested FAbs were purified by SP/AKTA using 50 mM NaOAc, pH5 with gradient NaCl elution (using 50 mM NaOAc + 1M NaCl, pH5).
    pH5
    suggested: None
    The plasmids were added to D10 medium in the following ratios: 10 µg pHAGE-Luc2-IRS-ZsGreen, 3.4 µg FL Spike, 2.2 µg HDM-Hgpm2, 2.2 µg HDM-Tat1b, 2.2 µg pRC-CMV-Rev1b in a final volume of 1000 µL.
    pRC-CMV-Rev1b
    suggested: RRID:Addgene_164443)
    Software and Algorithms
    SentencesResources
    The sequence alignment was again produced using MUSCLE31 and sequence identity was calculated using Geneious
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    The sequences used were: 6VXX, UniRef90_U5NJG5, _L7UP8, _A0A7U3W1C7, _K9N5Q8, _A0A2I6PIW5, _A0A3Q8AKM0, _U5WHZ7, _A0A5H2WTJ3, _A0A0U1WJY8, _A0A166ZND9, _A0A678TRJ7, _A0A2R4KP93, _A0A2Z4EVK1, _A0A7R6WCE7, _E0ZN36, _A0A6M3G9R1, _F1DAZ9, _A0A0U1UYX4, _A0A2R3SUW7, _A0A2Z4EVN5, _A0A2Z4EVN2, _U5LMM7, _A0A5Q0TVR4, _E0XIZ3, _A0A023Y9K3, _A0A2R4KP86, _A0A088DJY6, _A0A7G6UAJ9, _S4X276, _A0A4Y6GL90, _A3EXG6, _F1BYL9, _E0ZN60, _A0A0K1Z054, _A0A0U1WHI2, and NCBI accession numbers: YP_009047204.1, QLR06867.1, AAK32191.1, AGZ48828.1, AAT84362.1, QHR63300.2, ABD75513.1, YP_003767.1.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    The sequences were first aligned using the MUSCLE algorithm, and then two phylogenetic trees were made, both using PhyML 3.3.20180621.
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    PhyML
    suggested: (PhyML, RRID:SCR_014629)
    The sequences were then analyzed by sequence alignment using SnapGene software.
    SnapGene
    suggested: (SnapGene, RRID:SCR_015052)
    FAb Production from IgGs: 1/10 volume of 1M Tris, pH 8 was added to IgGs at ∼2 mg/mL in PBS. 2 µL of a 1 mg/mL stock of Lys-C (stock stored at -70C) was added for each mg of human IgG1 and digested for 1 hour at 37 °C with moderate rotation.
    IgGs
    suggested: None
    Final data analysis was done in Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    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.

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  2. Version published to 10.1101/2022.01.24.477625v1 on bioRxiv