A novel plant-made monoclonal antibody enhances the synergetic potency of an antibody cocktail against the SARS-CoV-2 Omicron variant

Abstract

This study describes a novel, neutralizing monoclonal antibody (mAb), 11D7, discovered by mouse immunization and hybridoma generation, against the parental Wuhan-Hu-1 RBD of SARS-CoV-2. We further developed this mAb into a chimeric human IgG and recombinantly expressed it in plants to produce a mAb with human-like, highly homogenous N-linked glycans that has potential to impart greater potency and safety as a therapeutic. The epitope of 11D7 was mapped by competitive binding with well-characterized mAbs, suggesting that it is a Class 4 RBD-binding mAb that binds to the RBD outside the ACE2 binding site. Of note, 11D7 maintains recognition against the B.1.1.529 (Omicron) RBD, as well neutralizing activity. We also provide evidence that this novel mAb may be useful in providing additional synergy to established antibody cocktails, such as Evusheld™ containing the antibodies tixagevimab and cilgavimab, against the Omicron variant. Taken together, 11D7 is a unique mAb that neutralizes SARS-CoV-2 through a mechanism that is not typical among developed therapeutic mAbs and by being produced in ΔXFT Nicotiana benthamiana plants, highlights the potential of plants to be an economic and safety-friendly alternative platform for generating mAbs to address the evolving SARS-CoV-2 crisis.

Keywords: Antibody cocktail; COVID-19; Monoclonal antibody (mAb); Neutralization synergy; Plant-made antibody; Variants of Concern.

Figure 1

Recombinant expression of 11D7 in ΔXFT Nicotiana benthamiana. ΔXTF N. benthamiana leaves were infiltrated with 11D7 gene constructs and total soluble leaf proteins were extracted 3–9 days post agroinfiltration (DPI). The expression levels of p11D7 were analysed by a sandwich ELISA that detects only fully assembled IgG. Mean ± SEM are shown from two independent experiments performed in technical duplicates.

Figure 2

Biochemical characterization of p11D7. Protein A‐purified, p11D7 and an IgG isotype control were subjected to SDS‐PAGE under reducing (a, Lanes 1 and 2) or non‐reducing conditions (a, Lanes 3 and 4) and total protein content was stained with Coomassie blue. In parallel, SDS‐PAGE‐separated proteins under non‐reducing (b) or reducing conditions (c and d) were transferred to PVDF and probed for human kappa light (b and c) or for human gamma chain (d). Lanes 1 and 3: IgG isotype control. Lanes 2 and 4: p11D7. HC: heavy chain. LC: light chain. (HL)₂: assembled heterotetrameric form of IgG. One representative result of multiple experiments is shown.

Figure 3

p11D7 recognition of RBD from various SARS‐CoV‐2 variants. Various dilutions of p11D7 and reference mAbs were incubated with the WA1/2020 RBD (a) or the B.1.1.529 (Omicron) RBD (b) that was immobilized on ELISA plates. Specific binding of the RBD‐mAb complex was detected with a horseradish peroxidase (HRP)—conjugated secondary antibody. The absorbance at 450 nm is plotted and is representative of at least two independent experiments performed with technical duplicates. Error bars represent SEM.

Figure 4

Neutralization of SARS‐CoV‐2 variants by p11D7. Serial dilutions of p11D7 were mixed with SARS‐CoV‐2 WA1/2020 (a), Delta (b), or Omicron (c) before adding to Vero E6 (a and b) or Vero‐hACE2‐TMPRSS2 (c) cells. Cells were then fixed, permeabilized and stained for SARS‐CoV‐2 spike protein (a and b) or nucleocapsid protein (c). Foci were counted, per cent neutralization determined, and IC₅₀ calculated using GraphPad Prism 9. Error bars are SD and curves represent at least two independent experiments performed in technical triplicates.

Figure 5

Competitive binding between p11D7 and other anti‐RBD mAbs to the WA1/2020 RBD. Dilutions of each reference mAb were coated on 96‐well plates followed by addition of 2 μg/mL of the WA1/2020 RBD. After washing, the plates were incubated with HRP‐conjugated p11D7 to detect either non‐competitive binding (detection of a signal) or overlapping binding (absence of a signal) of p11D7 with each reference mAb to RBD. Error bars represent SD and at least two independent experiments with technical triplicates were performed.