SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

Abstract

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

Fig. 1. Use of V region genes in human anti-SARS-CoV-2 antibodies.

Comparison of the distribution of V region gene subfamilies in the universal HAL9/10 library, the in vivo distribution of subfamilies, and the distribution of antibodies against S1 selected from HAL9/10. A Abundance of VH, B Vκ, and C Vλ.

Fig. 2. Inhibition of SARS-CoV-2 spike protein binding to cell (flow cytometry).

A Inhibition prescreen of 109 scFv-Fc antibodies on ACE2-positive cells using 1500 nM antibody and 50 nM spike protein (30:1 ratio). The antibodies selected for detailed analysis are marked in colors. Data show single measurements. B IC50 determination by flow cytometry using 50 nM S1-S2 trimer and 4.7–1500 nM scFv-Fc. C IC50 determination by flow cytometry using 10 nM RBD and 0.03–1000 nM scFv-Fc. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.

Fig. 3. Determination of EC50 on RBD.

Binding in titration ELISA of the 17 best inhibiting scFv-Fc on RBD (fusion protein with murine Fc part), S1 (fusion protein with murine Fc part), or S1-S2 (fusion protein with His tag). Sequence SARS-CoV-2 (Gene bank QHD43416). An unrelated antibody with murine Fc part (TUN219-2C1), human HEK293 cell lysate, BSA, or lysozyme were used as controls. Experiments were performed in duplicate and mean values are given. EC50 were calculated with GraphPad Prism Version 6.1, fitting to a four-parameter logistic curve.

Fig. 4. SARS-CoV-2 neutralization in the scFv-Fc format.

Neutralization analysis using 250 pfu of SARS-CoV-2 in a CPE-based neutralization assay. A Cell monolayer occupancy at 4 days post infection in the absence of neutralizing antibodies was compared to uninfected control cells and median values were normalized as 0 and 100% occupancy, respectively. Histograms indicate medians of normalized monolayer occupancy in a neutralization assay using 1 µg/mL (~10 nM) antibody for each of the 17 tested antibodies. Data show the median from 4 or 6 replicates, the black dots indicate monolayer occupancy in individual assays, and the range is given for the maximum and minimum measurements. B Representative phase-contrast microscopic pictures of uninfected cells, cells infected in the absence of antibodies, in the presence of a poorly neutralizing scFv-Fc (STE73-2C2), or of a highly neutralizing scFv-Fc (STE73-6C8).

Fig. 5. Inhibition of RBD–ACE2 interaction by IgG.

A IC50 determination by flow cytometry using 50 nM S1-S2-His and 0.5-500 nM IgG. B IC50 determination by flow cytometry using 10 nM RBD-mFC and 0.1–100 nM IgG. Palivizumab was used as negative control. The inhibition assays were made as single titrations. Logistic5 fit of Origin was used to determine the IC50.

Fig. 6. Binding to RBD mutants, epitopes, and structure models.

A ELISA using STE73-2E9, -9G3, and -2G8 on S1-His with different RBD mutations. B Overview of the binding of STE73-2E9, -9G3, and -2G8 to different RBD mutations analyzed by ELISA, SPR, and protein array. Sequence SARS-CoV-2 (Gene bank QHD43416). ELISA experiments were performed in duplicate and mean values are given. C The three antibodies STE73-2E9, -9G3, and -2G8 are binding to the ACE–RBD interface (docking models based on epitope data from binding to RBD mutations). Experimentally validated computational models of the variable regions of the antibodies (colored cartoons) binding to the RBD (white surface, same orientation in all images) are shown. The cartoon representation of ACE2 is also shown for comparison.

Fig. 7. Characterization of the neutralizing antibody STE73-2E9 in IgG format.

A Neutralization of 20–30 pfu SARS-CoV-2 by STE73-2E9, -9G3, and -2G8. Palivizumab was used as isotype control. B Validation of neutralization potency of STE73-2E9 using 100 pfu. Neutralization assays were performed in triplicates, mean ± s.e.m. are given. C Titration ELISA on the indicated antigens. ELISA shows single titration of two representative experiments (see also Supplementary Fig. 7). D Cross-reactivity to other coronavirus spike proteins analzyed by ELISA. S1-HIS SARS-CoV-2 Hi5 was produced in house. S1-HIS SARS-CoV-2 HEK and all other coronavirus S1 domain proteins were obtained commercially. ELISA experiments were performed in duplicate and the mean values are given. E, F Kinetic parameter determination through single-cycle kinetic titration SPR of STE73-2E9 IgG on HEK cell produced RBD-SD1 and S1-S2, respectively (concentrations: 200, 100, 50, 25, 12.5, 6.25 nM).