A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern

Abstract

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable in overcoming the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab_35B5-47D10, which successfully retained parental specificity and simultaneously bound to the two distinct epitopes on receptor-binding domain (RBD) and S2. Bi-Nab_35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs), and variants being monitored (VBMs) compared with its parental monoclonal antibodies (MAbs). Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab_35B5-47D10 can efficiently neutralize VBMs, including Alpha (B.1.1.7), Beta (B.1.351), and Kappa (B.1.617.1), as well as VOCs, including Delta (B.1.617.2), Omicron BA.1, and Omicron BA.2. Crucially, Bi-Nab_35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC₅₀ = 0.15 nM) and Omicron BA.2 (IC₅₀ = 0.67 nM) compared with its parental MAbs. Therefore, Bi-Nab_35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. IMPORTANCE The new, highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab_35B5-47D10, which simultaneously targets SARS-CoV-2 RBD and S2 and improves the neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab_35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab_35B5-47D10 is a feasible and potentially effective strategy by which to treat and prevent COVID-19.

Keywords: COVID-19; Omicron variants; SARS-CoV-2; bispecific antibodies; neutralization; neutralizing antibodies.

FIG 1

Construction and generation of bsAbs. (A and B) Overview of the strategy for designing bsAbs. Schematic diagram of the molecular configurations of Bi-Nab_35B5-47D10 (A) and Bi-Nab_47D10-35B5 (B). (C) Schematic presentation of the bsAbs. Antibody domains are color-coded according to their architecture (green, variable light chain of 35B5; red, variable heavy chain of 35B5; blue, variable light chain of 47D10; yellow, variable heavy chain of 47D10; gray, human IgG1 Fc). (D) Reduced SDS-PAGE analysis of two bsAbs and two parental MAbs. The proteins were analyzed under reducing conditions (+DTT). H and L denote the heavy and light chains, respectively. The molecular weight of the bsAbs monomer was 90 kDa. M, molecular weight standard. (E) Nonreduced SDS-PAGE analysis of affinity purified bsAbs and parental MAbs. All four antibodies were expressed in ExpiCHO-S cells and captured on protein A affinity resin. The proteins were analyzed under direct affinity elution conditions (without DTT). Three independent experiments were performed at this scale and yielded the same results. Additional independent experiments yielded similar results at larger culture volumes.

FIG 2

Binding and inhibition properties of bsAbs. (A and B) ELISA binding assay of bsAbs and parental MAbs to the S1 protein (A) or the S2 protein (B) of WT SARS-CoV-2. EC₅₀, concentration for 50% of maximal effect. (C) ELISA analysis of bsAbs or a cocktail or parental MAb-mediated inhibition of WT RBD protein binding to ACE2. For the antibody cocktail, the value on the x axis refers to the final antibody concentration. IC₅₀, half maximal inhibitory concentration. (D) ELISA analysis of bsAbs or parental MAbs binding to the mutated S1 protein of SARS-CoV-2, including HV69-70 deletion, N501Y, and D614G. (E) Representative EC₅₀ and IC₅₀ titers of bsAbs and parental MAbs showing the effective binding and inhibiting activity of Bi-Nab_35B5-47D10.

FIG 3

Binding kinetics of bsAbs to cell surface-associated coronavirus S protein. (A to E) Binding of bsAbs or parental MAbs to RaTG13 S, WT SARS-CoV-2 S, Alpha S, Beta S, or Delta S proteins. HEK293T cells were transfected to transiently express RaTG13 S, WT SARS-CoV-2, Alpha S, Beta S, or Delta S proteins and were incubated with MAb 35B5 (B), MAb 47D10 (C), Bi-Nab_35B5-47D10 (D), and Bi-Nab_47D10-35B5 (E), respectively, for 1 h on ice. Soluble hACE2 with a His tag was used as a positive-control (A), followed by a FITC-conjugated anti-human IgG Fc antibody or a FITC-conjugated anti-His antibody. Then, the cells were analyzed by flow cytometry. Mean fluorescence intensity (MFI) was normalized to the empty vector (mock) group (F). The experiments were performed three times, and one representative is shown.

FIG 4

RBD and S2 mutations of VBM variants. (A) Statistics on VBM RBD and S2 mutations are displayed. (B) The crystal structure of the SARS-CoV-2 spike trimer (PDB ID: 7KRQ), highlighting the mutational landscape of VBM variants relative to WT SARS-CoV-2. The epitopes of the RBD (bright blue) and S2 (dark red) regions are shown. The mutations are indicated by yellow (RBD mutations) and green (S2 mutations) spheres on the surface of the S trimer, using the PyMOL software suite.

FIG 5

Neutralization of bsAbs against WT and VBM pseudoviruses. (A to D) Two parental MAbs and two bsAbs mediated neutralization of the indicated pseudovirus. WT SARS-CoV-2 S (A), Alpha S (B), Beta S (C), or Kappa S (D) pseudovirus were preincubated with 5-fold serially diluted Bi-Nab_47D10-35B5, Bi-Nab_35B5-47D10, 47D10, 35B5. Then, the mixture was added to HEK293 cells transiently expressing hACE2 and lysed 48 h later, and their transduction was measured according to luciferase activities. Potencies were calculated against sensitive viruses, and heatmaps of IC₅₀ titers were generated in Excel. Warmer colors indicate more potent neutralization. Breadths based on IC₅₀s are also summarized (E). Representative IC₅₀ titers and neutralization breadths of bsAbs and the parental MAbs showing the improved neutralization activity of Bi-Nab_35B5-47D10. NE, no effect. The experiment was performed twice, and one representative is shown. Error bars represent the standard error of the mean of technical triplicates.

FIG 6

RBD and S2 mutations of VOC variants. (A) Schematic of VOC RBD and S2 mutations is illustrated. (B) Top view (left panel) and side view (right panel) of spike trimer are shown with mutations in RBD and S2 and highlighted with residue atoms as colored spheres, indicated in yellow (RBD mutations, the red font indicates the mutations unique to the Omicron variant, and the pink refers to the mutations common to VOCs) and green (S2 mutations) on the surface of the S trimer (PDB ID: 7KRQ).

FIG 7

Cross-reactive neutralization of bsAbs against Delta and Omicron pseudoviruses. Using a lentiviral-based pseudovirus system, the neutralization potency of two parental MAbs and two bsAbs against Delta (A), Omicron BA.1 (B), and Omicron BA.2 (C) pseudoviruses were analyzed. The IC₅₀ was determined by the log (inhibitor) response of a nonlinear regression analysis, and bars and error bars depict the mean and the standard error of the mean, respectively. (D) Bi-Nab_35B5-47D10 exhibited significantly improved neutralization activity compared to Bi-Nab_47D10-35B5 and parental MAbs. IC₅₀ titers, breadth, and potency of two bsAbs and two parental MAbs against Delta and Omicron pseudoviruses are presented with heat maps. NE, no effect.