Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach
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Abstract
SARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naïve phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.
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SciScore for 10.1101/2020.09.27.316174: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were conducted according to the National Institutes of Health guidelines. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Hamster challenge experiments: Male and female Syrian golden hamsters were obtained from Charles River Laboratories at 6 weeks of age. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibody binding ELISA: The S1 subunit of the SARS-CoV-2 spike protein (amino acids 16-685) bearing a C-terminal histidine tag … SciScore for 10.1101/2020.09.27.316174: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were conducted according to the National Institutes of Health guidelines. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable Hamster challenge experiments: Male and female Syrian golden hamsters were obtained from Charles River Laboratories at 6 weeks of age. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibody binding ELISA: The S1 subunit of the SARS-CoV-2 spike protein (amino acids 16-685) bearing a C-terminal histidine tag (ACRO Biosystems, Newark, NJ) was coated at 2 μg/ml on a Ni-NTA plate (Qiagen, Valencia, CA). SARS-CoV-2 spike protein (amino acids 16-685suggested: NoneAntibody characterization: Kinetic interactions between the antibodies and His-tagged receptor binding domain (RBD, amino acids 319-537) (Acro Biosystems, Newark, NJ) protein was measured at 25°C using BIAcore T200 surface plasmon resonance (SPR) (GE Healthcare). His-tagged receptor binding domain (RBD, amino acids 319-537) (Acro Biosystems, Newark, NJsuggested: NoneSTI-1499 or STI-2020 antibody was covalently immobilized on a CM5 sensor chip to approximately 500 and 100 resonance units (RU), respectively using standard N-hydroxysuccinimide/N-Ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (NHS/EDC) coupling methodology. STI-2020suggested: NoneFor antibody binding to the cells expressing the Spike proteins, the cells were dispensed into wells of a 96-well plate (25 μl per well), and an equal volume of 2x final concentration of serially-diluted anti-S1 antibody solution was added. anti-S1suggested: NoneAntibody treatments were administered intravenously (i.v.) with monoclonal antibodies (mAbs) against SARS-CoV-2 Spike, or isotype control mAb in up to 350 µl of sterile PBS at 1 hour-post inoculation. SARS-CoV-2suggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293 cells were transfected using FuGeneHD transfection reagent according to manufacturer’s protocol (Promega, Cat # E2311). HEK293suggested: NoneVero E6 cells were plated to 96-well plates and incubated at 37° C, 5% CO2 Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Cell based Spike binding assay: Mammalian expression vectors were constructed either by cloning of the synthesized gene encoding SARS-CoV-2 G614 Spike protein (UniprotKB, SPIKE-SARS2) or, for SARS-CoV-2 D614 Spike protein, via site-directed mutagenesis of the G614 Spike protein gene. UniprotKBsuggested: (UniProtKB, RRID:SCR_004426)A sigmoidal four-parameter logistic equation was used for fitting the MFI vs. mAb concentration data set to extract EC50 values (GraphPad Prism 8.3.0 software). GraphPad Prismsuggested: (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.
- No funding statement was detected.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.09.27.316174: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were conducted according to the National Institutes of Health guidelines. Randomization 94 single clones (black dots) were randomly selected and together with 2 clones representing wild type STI-1499 as reference (red dots) were analyzed by ELISA. Blinding not detected. Power Analysis not detected. Sex as a biological variable Hamster challenge experiments Male and female Syrian golden hamsters were obtained from Charles River Laboratories at 6 weeks of age. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences R… SciScore for 10.1101/2020.09.27.316174: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Medical Branch and were conducted according to the National Institutes of Health guidelines. Randomization 94 single clones (black dots) were randomly selected and together with 2 clones representing wild type STI-1499 as reference (red dots) were analyzed by ELISA. Blinding not detected. Power Analysis not detected. Sex as a biological variable Hamster challenge experiments Male and female Syrian golden hamsters were obtained from Charles River Laboratories at 6 weeks of age. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibody characterization Kinetic interactions between the antibodies and His-tagged receptor binding domain (RBD, amino acids 319-537) (Acro Biosystems, Newark, NJ) protein was measured at 25C using BIAcore T200 surface plasmon resonance (SPR) (GE Healthcare). His-tagged receptor binding domain (RBD, amino acids 319-537) (Acro Biosystems, Newark, NJsuggested: NoneExpression and purification of STI-1499 and STI-2020 monoclonal antibodies STI-1499 was expressed using a two-vector transient expression protocol. STI-2020suggested: NoneFor antibody binding to the cells expressing the Spike proteins, the cells were dispensed into wells of a 96-well plate (25 l per well), and an equal volume of 2x final concentration of serially-diluted anti-S1 antibody solution was added. anti-S1suggested: NoneAntibody treatments were administered intravenously (i.v.) with monoclonal antibodies (mAbs) against SARS-CoV-2 Spike, or isotype control mAb in up to 350 µl of sterile PBS at 1 hour-post inoculation. SARS-CoV-2suggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293 cells were transfected using FuGeneHD transfection reagent according to manufacturer’s protocol (Promega, Cat # E2311). HEK293suggested: NoneVero E6 cells were plated to 96-well plates and incubated at 37° C, 5% CO2 Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Cell based Spike binding assay Mammalian expression vectors were constructed either by cloning of the synthesized gene encoding SARS-CoV-2 G614 Spike protein (UniprotKB, SPIKE-SARS2) or, for SARS-CoV-2 D614 Spike protein, via sitedirected mutagenesis of the G614 Spike protein gene. UniprotKBsuggested: (UniProtKB, RRID:SCR_004426)A sigmoidal four-parameter logistic equation was used for fitting the MFI vs. mAb concentration data set to extract EC50 values (GraphPad Prism 8.3.0 software). GraphPad Prismsuggested: (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.
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