Discovery and characterization of high-affinity, potent SARS-CoV-2 neutralizing antibodies via single B cell screening

Abstract

Monoclonal antibodies that target SARS-CoV-2 with high affinity are valuable for a wide range of biomedical applications involving novel coronavirus disease (COVID-19) diagnosis, treatment, and prophylactic intervention. Strategies for the rapid and reliable isolation of these antibodies, especially potent neutralizing antibodies, are critical toward improved COVID-19 response and informed future response to emergent infectious diseases. In this study, single B cell screening was used to interrogate antibody repertoires of immunized mice and isolate antigen-specific IgG1⁺ memory B cells. Using these methods, high-affinity, potent neutralizing antibodies were identified that target the receptor-binding domain of SARS-CoV-2. Further engineering of the identified molecules to increase valency resulted in enhanced neutralizing activity. Mechanistic investigation revealed that these antibodies compete with ACE2 for binding to the receptor-binding domain of SARS-CoV-2. These antibodies may warrant further development for urgent COVID-19 applications. Overall, these results highlight the potential of single B cell screening for the rapid and reliable identification of high-affinity, potent neutralizing antibodies for infectious disease applications.

Figure 1

Overview of methodologies for discovery and characterization of potent SARS-CoV-2 neutralizing antibodies isolated from single B cells. (1) BALB/c mice were immunized with SARS-CoV-2 RBD three times at 2-week intervals. (2) Splenocytes were harvested six weeks following the initial vaccination from which B cells were isolated via MACS and labeled for cell sorting. (3) Cells positive for binding to SARS-CoV-2 RBD labeled with two unique fluorophores (gate R7) were sorted as single cells into 96-well plates. (4) Multiple PCR steps were employed to isolate V_H and V_L antibody genes. (5) Antibodies were expressed, purified, and evaluated for antigen binding, neutralizing activity, and biophysical properties.

Figure 2

RBD-immunized mice demonstrate significant total RBD-specific IgG and IgG1 responses. (A) BALB/c mice were vaccinated three times at 2-week intervals. On weeks 4, 6, and 10, serum samples were collected for total IgG (B) and IgG1 (C) titer measurements using a RBD-specific ELISA. A subset of mice was used to harvest spleens for B cell isolation on week 6. Data are presented as mean of reciprocal EC₅₀ ± SEM. **p < 0.01, ***p < 0.001, and ****p < 0.0001 by two-way ANOVA followed by row comparisons.

Figure 3

Gating strategy for the isolation of RBD-specific memory B cells via single cell sorting. Flow cytograms of cell samples from (A–G) immunized BALB/c mice and (H) control naïve mice are shown. The following (A–G) gating strategy was applied in series and gate R7 was sorted as single cells into 96-well plates: (A) selection of lymphocytes based on size (forward scatter area) and granularity (side scatter area), (B) doublet discrimination based on forward scatter width and forward scatter area, (C) viability and lack of non-B cell markers (T cell marker CD4, T cell marker CD8, neutrophil marker GR-1, macrophage marker F480), (D) B cell marker CD19, (E) negative for IgM (naïve B cell marker) and positive for IgG1, (F) negative for IgD (naïve B cell marker), and (G) binding to SARS-CoV-2 RBD labeled with PE and APC fluorescent proteins. Flow cytograms for control naïve mice are presented in Supplementary Fig. 1. The SARS-CoV-2 RBD antigen binding cytogram for (H) naïve mice is presented for comparison.

Figure 4

Isolated antibodies possess high affinity for the SARS-CoV-2 receptor-binding domain. Flow cytometry analysis of binding of antibodies to biotinylated SARS-CoV-2 RBD immobilized on magnetic beads was assessed via flow cytometry. Half-maximal effective concentrations (EC₅₀) are presented for the high affinity antibodies (12H2, 13I1) and a positive control antibody (CB6). The results are averages of three independent experiments and the error bars are standard deviations.

Figure 5

Isolated antibodies demonstrate potent neutralizing activity in a SARS-CoV-2 pseudovirus assay. Neutralizing activities of the high affinity antibodies (12H2 and 13I1) are shown relative to a potent neutralizing control antibody (CB6) and two control antibodies with moderate (VHH-72) and weak (CR3022) neutralizing activity. A lentivirus-based SARS-CoV-2 pseudovirus assay was employed, and the relative luciferase signal was measured 48 h post infection. Half-maximal inhibitory concentrations (IC₅₀) values are reported. These data are averages of three repeats, and the error bars are standard deviations.

Figure 6

Neutralizing antibody 13I1 competes with ACE2 for binding to the SARS-CoV-2 receptor-binding domain. (A) Schematic of competition analysis between 13I1, ACE2 and other antibodies (CR3022, CB6, VHH-72, C119, S309). Competitive binding analysis was employed in which 13I1, reference antibodies or ACE2 was pre-incubated with biotinylated RBD (5 nM) over a range of concentrations of antibodies or ACE2, and then co-incubated with immobilized 13I1 on Protein A magnetic beads. (B) The percentage of SARS-CoV-2 RBD bound is reported relative to the amount bound in the absence of pre-blocking with antibodies or ACE2. The results are averages from two independent experiments and the error bars are standard deviations.

Figure 7

Increased valency enhances 13I1 neutralizing activity. Neutralizing activity of tetravalent 13I1 DVD-formatted antibody is shown relative to 13I1 bivalent antibody. A lentivirus-based SARS-CoV-2 pseudovirus assay was employed, and the relative luciferase signal was measured 48 h post infection. Calculated half-maximal inhibitory doses (IC₅₀) are presented. 13I1 DVD neutralizing activity was evaluated in parallel with 13I1 data collected in Fig. 5 and is shown here for clarity. These data are averages of three independent repeats, and the error bars are standard deviations.

Figure 8

Neutralizing antibodies possess high specificity and stability. (A) Non-specific binding of antibodies (immobilized on Protein A magnetic beads) was evaluated via incubation with biotinylated soluble membrane proteins from CHO cells and detection via flow cytometry. Control antibodies with high (emibetuzumab) and low (elotuzumab) non-specific binding were also evaluated for comparison. The two control antibodies are not identical to the actual drugs, as they have the variable regions of the actual drugs and a common IgG1 framework. (B) Melting temperatures of antibodies were evaluated via differential scanning fluorimetry. Results are averages from two independent experiments and the error bars are standard deviations.