A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to most known recently emerging RBD mutations

Abstract

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed an IC50 of 0.56 nM in a plaque-based live SARS-CoV-2 neutralization assay. The crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD was solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. In contrast to other published anti-SARS-CoV-2 antibodies, the binding of STE90-C11 is not blocked by known RBD mutations, endowing our antibody with higher intrinsic resistance to those possible escape mutants.

Identification of cell lines CL-14, CL-40 and CAL-51 as suitable models for SARS-CoV-2 infection studies

“…However, it is unclear whether different levels of ACE2 expression on the surface of cells would interfere with the ability to block virus entry. Thus, we used three different permissive cell lines of our panel (CL-14, CAL-51, and VERO-E6) with varying ACE2 levels and performed in vitro neutralisation assays using the therapeutic anti-Spike antibody COR-101 currently used in clinical trials (see S3 Fig ) [ 48 ]. While the comparability among these cell lines is limited due to several variables (cell density, virus growth kinetic, cell medium, etc.…”

Sequence signatures of two IGHV3-53/3-66 public clonotypes to SARS-CoV-2 receptor binding domain

“…Interestingly, a Y58F mutation results in a loss of hydrogen bonding interactions between residue 58 of the heavy chain and T415 of the RBD ( Supplementary Figure 10), yet the mutation significantly increases the binding affinity of the antibody to the RBD. We then performed a structural analysis on seven IGHV3-53/66 RBD antibodies with Y58F mutation and nine without 26,29,38,40,47,[54][55][56][57] . Our results indicate that, by removal of the hydroxyl group, the side chain of Y58F moves closer to the backbone carbon of RBD T415 (Supplementary Figure 10).…”

The N501Y and K417N mutations in the spike protein of SARS-CoV-2 alter the interactions with both hACE2 and human derived antibody: A Free energy of perturbation study

“…After the solvation with SPC waters the complex consisted of over 102 000 atoms. For the study of S1 RBD interactions with the neutralizing antibody STE90-C11 selected from COVID-19 patients [14], which was well tolerated to the known mutations, we use the very recently published X-ray structure with a PDB access number of 7B3O. For the FEP calculations with double N501Y/K417N mutations we used as a starting point the energy minimized most representative frame of the N501Y FEP simulation.…”

“…This is a significant value and the observed convergence was good ( Table 1 and Figures 1B and 1E). The binding energy of the antibody can be roughly estimated based on the published value of IC50=0.56nM in a plaque-based live SARS-CoV-2 neutralization assay [14]. This is equivalent to ΔG value of about -12.7 kcal/mol (ΔG=RTln (IC50).…”