Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library

Abstract

SARS-CoV-2 (Covid-19) has caused currently ongoing global plague and imposed great challenges to health managing systems all over the world, with millions of infections and hundreds of thousands of deaths. In addition to racing to develop vaccines, neutralizing antibodies (nAbs) to this virus have been extensively sought and are expected to provide another prevention and therapy tool against this frantic pandemic. To offer fast isolation and shortened early development, a large human naïve phage display antibody library, was built and used to screen specific nAbs to the receptor-binding domain, RBD, the key for Covid-19 virus entry through a human receptor, ACE2. The obtained RBD-specific antibodies were characterized by epitope mapping, FACS and neutralization assay. Some of the antibodies demonstrated spike-neutralizing property and ACE2-competitiveness. Our work proved that RBD-specific neutralizing binders from human naïve antibody phage display library are promising candidates to for further Covid-19 therapeutics development.

SciScore for 10.1101/2020.05.19.104281: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Alternatively, 10 μg/ml antibody A was captured by anti-human Fc biosensor (ForteBio, cat# 18-5060) until saturation, followed by sequential binding of 10 μg/ml RBD, antibody B.	anti-human Fc biosensor suggested: None
To examine if the antibody hits from ELISA screening were still capable of binding to native spike trimer, the individual antibody hit at 5 μg/ml and/or ACE2-mFc was incubated with 1-2 million 1D8 spike+ cells and detected by anti-human-Fc PE …

SciScore for 10.1101/2020.05.19.104281: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Alternatively, 10 μg/ml antibody A was captured by anti-human Fc biosensor (ForteBio, cat# 18-5060) until saturation, followed by sequential binding of 10 μg/ml RBD, antibody B.	anti-human Fc biosensor suggested: None
To examine if the antibody hits from ELISA screening were still capable of binding to native spike trimer, the individual antibody hit at 5 μg/ml and/or ACE2-mFc was incubated with 1-2 million 1D8 spike+ cells and detected by anti-human-Fc PE (invitrogen, 12-4998-82) and FITC Goat anti-mouse-IgG (Biolegend, cat#405305) or APC Goat anti-mouse-IgG (Biolegend, cat# 405308) following standard FACS protocol and analyzed by Beckman FACS (CytoFLEX, CytExpert2.0).	anti-human-Fc PE suggested: None anti-mouse-IgG suggested: (Proteintech Cat# 10283-1-AP, RRID:AB_2877728)
Neutralization assay of RBD-antibodies by FACS: To check whether ACE2-Spike interaction can be blocked by the isolated nAbs, individual titrated antibody from 10 ug/ml down to 0.31 ug/ml (2 times down) was incubated with 1-2 spike+ cells for 1 hour at 4°C before adding of 0.02 ug/ml ACE2-mFc followed by FITC conjugated anti-mouse IgG (Biolegend, cat#405305) and the MFI of FITC and positive percentage were collected.	anti-mouse IgG suggested: (BioLegend Cat# 405305, RRID:AB_315008)
Experimental Models: Cell Lines
Sentences	Resources
Construction of Covid-19 spike-expressing cell line: To express the spike (S) protein of SARS-CoV-2 in mammalian cells, a codon optimized cDNA (GenBank:NC_045512.2) encoding the full-length S protein, a transmembrane motif (TM) and 3xFLAG tag was synthesized and cloned into a pRRL-derived vector (R48) by XbaI/XmaI, yielding pRRL-19S-FLAG-BSD. 293FT cells were transfected with lenti-viral made from SARS-CoV-2 S plasmid and other vectors (pLP-1-Gag/Pol, pLP-2-Rev and pLP-VSVG, all in-house made) containing packaging elements.	293FT suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
One each of Kappa and lambda EHL Library aliquots were mixed and blocked with 4% milk PBS (MPBS), 10 million 293F cells (to deplete potential binders to residual protein contaminated in vendor’s RBD) and 10μg/ml mouse IgG (to deplete mouse Fc binders) before adding the library stock to the RBD-coating wells.	293F suggested: None

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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Isolation of and Characterization of Neutralizing Antibodies to Covid-19 from a Large Human Naïve scFv Phage Display Library

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