An Engineered Antibody with Broad Protective Efficacy in Murine Models of SARS and COVID-19

Article activity feed

1. 

   ScreenIT

   Nov 18, 2020

   SciScore for 10.1101/2020.11.17.385500: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Fixed and permeabilized cells were first stained with a primary antibody recognizing SARS-CoV nucleocapsid protein (Sino Biological), followed by secondary antibody staining with AlexaFluor 488-conjugated goat anti-rabbit antibody.	SARS-CoV nucleocapsid protein suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503) anti-rabbit suggested: None
   Virus and antibody mixture was then added to Vero E6 cells and incubated at 37 °C with 5% CO2 for 48 hours (SARS-CoV-MA15, SARS-CoV-2-MA2 and SARS-CoV-2-nLuc) or 24 hours (SHC014-nLuc and WIV1-nLuc).	SARS-CoV-MA15 suggested: None SARS-CoV-2-MA2 suggested: None SAR…

   SciScore for 10.1101/2020.11.17.385500: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Fixed and permeabilized cells were first stained with a primary antibody recognizing SARS-CoV nucleocapsid protein (Sino Biological), followed by secondary antibody staining with AlexaFluor 488-conjugated goat anti-rabbit antibody.	SARS-CoV nucleocapsid protein suggested: (Creative Diagnostics Cat# DMAB8869, RRID:AB_2392503) anti-rabbit suggested: None
   Virus and antibody mixture was then added to Vero E6 cells and incubated at 37 °C with 5% CO2 for 48 hours (SARS-CoV-MA15, SARS-CoV-2-MA2 and SARS-CoV-2-nLuc) or 24 hours (SHC014-nLuc and WIV1-nLuc).	SARS-CoV-MA15 suggested: None SARS-CoV-2-MA2 suggested: None SARS-CoV-2-nLuc suggested: None
   SARS-CoV-2 viruses were detected with a mixture of CC6.29, CC6.33, L25-dP06E11, CC12.23, and CC12.25 antibodies, previously derived from a cohort of convalescent SARS-CoV-2 donors (23)	CC12.25 suggested: None
   In vitro affinity maturation of ADI-55688, ADI-55689, and ADI-56046: For each antibody, the complementarity-determining regions (CDRs) 1, 2, and 3 of the heavy- and light-chains were diversified separately via oligo-based mutagenesis using NNK-randomized oligos spanning CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 (IDT).	CDRH2 suggested: None CDRH3 suggested: None CDRL1 suggested: None CDRL2 suggested: None CDRL3 suggested: None
   Antibody-dependent natural killer cell activation and degranulation (ADNKDA): Primary human NK cells were enriched from the peripheral blood of human donors using RosetteSep Human NK cell Enrichment Cocktail (Stem Cell Technologies, Cat #15065) and cultured overnight in RPMI-1640 (Corning, Cat # 15-040-CV) supplemented with 10% FBS (Hyclone, Cat # SH30071.03), 1% Pen/Strep (Gibco, Cat # 15070-063), 1% L-Glutamine (Corning, Cat # 25-005-CI), 1% HEPES (Corning, Cat # 25-060-CI) and 5 ng/mL recombinant human IL-15 (StemCell Technologies, Cat # 78031)	25-060-CI suggested: None
   Unbound antibodies were removed by washing with PBS were added at 5 × 104 cells/well in the presence of 4 μg/mL brefeldin A (Biolegend, Cat # 420601), 5 μg/mL GolgiStop (BD Biosciences, Cat # 554724) and anti-CD107a antibody (Clone H4A3 PE-Cy7, Biolegend, Cat # 328618) for 5 hours.	anti-CD107a suggested: (BioLegend Cat# 328618, RRID:AB_11147955)
   Antibody-dependent cellular phagocytosis (ADCP) with monocytes and neutrophils: For ADCP assays with neutrophils, HL-60 promyeloblast cells (ATCC, Cat # CCL-240) were maintained in Iscove’s Modified Dulbecco’s Medium (ATCC, Cat # 30-2005) with 20% fetal bovine serum and 1% Pen/Strep.	Antibody-dependent cellular phagocytosis ( ADCP suggested: None
   Beads were washed with PBS containing 15 mM EDTA, and stained with an FITC-conjugated anti-guinea pig C3 antibody (MP Biomedicals, Cat # 855385).	anti-guinea pig C3 suggested: None
   Briefly, polyclonal goat anti-human IgG Fc antibodies (capture; Jackson ImmunoResearch Laboratories) and polyclonal goat non-specific antibodies (non-capture; Jackson ImmunoResearch Laboratories) were buffer exchanged into 20 mM sodium acetate (pH 4.3) and concentrated to 0.4 mg/mL.	anti-human IgG suggested: None
   Antibody binding to yeast surface-displayed RBD variants: To assess binding breadth, IgGs and recombinant hACE2 (expressed in a bivalent format as a C-terminal IgG1 Fc conjugate; Sino Biological, Cat # 10108-H02H) were tested against the panel of 17 sarbecovirus RBDs.	C-terminal IgG1 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   (hACE2)-expressing HeLa cells for authentic SARS-CoV-2 neutralization assays were generated as previously described (11).	HeLa suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
   To generate single-round infection competent pseudoviruses, HEK293T cells were co-transfected with 2 μg of MLV Gag-Pol-, 2 μg of MLV luciferase-, and 0.5 μg of either SARS-CoV-2 WT S or SARS-CoV-2 D614G S-encoding plasmids in 6-well plates using Lipofectamine 2000 (Thermo FisherScientific), according to the manufacturer’s directions.	HEK293T suggested: None
   Following infection for 42-48 hours at 37 °C, HeLa-hACE2 cells were lysed with 1x luciferase lysis buffer (25 mM Gly-Gly pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100)	HeLa-hACE2 suggested: None
   For neutralization assays, HeLa-hACE2 or Vero E6 target cells were seeded in a 96-well half-well plate at approximately 8000 cells/well suspended in 50 μL complete DMEM and grown overnight.	Vero E6 suggested: None
   ADG1-3 and benchmark SARS-CoV-2 mAbs REGN10933, REGN10987, CB6/LY-CoV016, and S309 were expressed in CHO cells as full-length IgG1 proteins.	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   HL-60 cells were differentiated into neutrophils by growth for 5 days in the presence of 1.3% DMSO.	HL-60 suggested: CLS Cat# 300209/p671_HL-60, RRID:CVCL_0002)
   Unbound antibodies were removed by centrifugation prior to the addition of THP-1 cells at 2.5 × 104 cells/well.	THP-1 suggested: None
   Briefly, soluble membrane protein (SMP) and soluble cytosolic protein (SCP) fractions were extracted from Chinese hamster ovary (CHO) cells and biotinylated using NHS-LC-Biotin (Thermo Fisher Scientific) reagent	Chinese hamster ovary ( CHO ) suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   ADG1-3 and benchmark SARS-CoV-2 mAbs REGN10933, REGN10987, CB6/LY-CoV016, and S309 were expressed in CHO cells as full-length IgG1 proteins.	CB6/LY-CoV016 suggested: None
   Animal studies: Twelve-month old female Balb/c mice (Envigo, strain 047) were treated with 200 μg of ADG-2 IgG via intraperitoneal (IP) injection at either 12 hours prior to infection (prophylactic) or 12 hours post-infection (therapeutic).	Balb/c suggested: None
   Software and Algorithms
   Sentences	Resources
   Data points were fit with a second-order polynomial in Excel to obtain wavelengths at maximum absorbance.	Excel suggested: None
   Multiple sequence alignment of sarbecovirus RBD sequences was visualized in Jalview.	Jalview suggested: (Jalview, RRID:SCR_006459)
   Mean anti-human IgG PE MFI signal was normalized according to the formula: (MFIsample – MFIminimum)*100/(1 – MFIminimum) and fitted as nonlinear regression curves in GraphPad Prism using the following equation: Y=Yx=minimum + X*(Yx=max – Yx=minimum)/(KDApp + X), where × is the IgG or hACE2 concentration and Y is the normalized binding signal.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Motion correction, CTF-estimation and particle picking were performed in Warp (46) and extracted particles were imported into cryoSPARC v2.15.0 (47).	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)

   ---

   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.

   ---

   Read the original source
2. 

   Version published to 10.1101/2020.11.17.385500v1 on bioRxiv

   Nov 17, 2020