Potential for a Plant-Made SARS-CoV-2 Neutralizing Monoclonal Antibody as a Synergetic Cocktail Component

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a public health crisis over the last two years. Monoclonal antibody (mAb)-based therapeutics against the spike (S) protein have been shown to be effective treatments for SARS-CoV-2 infection, especially the original viral strain. However, the current mAbs produced in mammalian cells are expensive and might be unaffordable for many. Furthermore, the emergence of variants of concern demands the development of strategies to prevent mutant escape from mAb treatment. Using a cocktail of mAbs that bind to complementary neutralizing epitopes is one such strategy. In this study, we use Nicotiana benthamiana plants in an effort to expedite the development of efficacious and affordable antibody cocktails against SARS-CoV-2. We show that two mAbs can be highly expressed in plants and are correctly assembled into IgG molecules. Moreover, they retain target epitope recognition and, more importantly, neutralize multiple SARS-CoV-2 variants. We also show that one plant-made mAb has neutralizing synergy with other mAbs that we developed in hybridomas. This is the first report of a plant-made mAb to be assessed as a potential component of a SARS-CoV-2 neutralizing cocktail. This work may offer a strategy for using plants to quickly develop mAb cocktail-based therapeutics against emerging viral diseases with high efficacy and low costs.

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

“…In an ELISA assay using the WA1/2020 RBD and p11D7 conjugated to horseradish peroxidase (HRP), we observed that CR3022 binding to the RBD abolished the ability of p11D7 to bind to the RBD, while several other EUA therapeutic mAbs (CB6, tixagevimab and cilgavimab) did not inhibit the binding of p11D7 to the RBD (Figure 5 ). This was the expected outcome for CR3022 and CB6, given that we have previously provided evidence of this (Jugler et al ., 2022 ), validating that p11D7 has at least a partially overlapping epitope on the RBD with CR3022 (a Class 4 mAb) but not with CB6 (also known as etesevimab, a Class 1 mAb). Similarly, the two mAbs, tixagevimab (also known as COV2‐2196, a Class 1/2 mAb) and cilgavimab (COV2‐2130, a Class 3 mAb) did not compete with the binding of p11D7 to the RBD.…”

“…The chimeric 11D7 transiently expressed in ΔXFT N. benthamiana plants peaked within a week after agroinfiltration to approximately 131 μg of p11D7 per gram of FLW. This level of expression is similar to other mAbs recently produced by our group using the same expression vector based on the geminiviral bean yellow dwarf virus (Jugler et al ., 2022 ) and allowed accumulation of milligram levels necessary for in vitro and future in vivo studies. However, this level of expression is lower than those of mAbs we produced using more optimized geminiviral vectors (Diamos et al ., 2020 ) or other plant‐made proteins such as griffithsin (GRFT) (Fuqua et al ., 2015 ) that have shown potent anti‐viral activity against SARS‐CoV‐2 (Alsaidi et al ., 2021 ; Palmer, 2022 ).…”

“…The correct assembly was further validated by observing specific binding of the mAb to its target antigen, the WA1/2020 RBD. The dissociation constant determined from the ELISA was 0.15 nM, suggesting that our novel mAb has a relatively high affinity for the SARS‐CoV‐2 RBD as this is similar to the dissociation constant for other neutralizing mAbs against SARS‐CoV‐2, CA1 and CB6, produced in plants (Jugler et al ., 2022 ). Interestingly, although the affinity is close to that of these neutralizing mAbs, the IC 50 for p11D7 in neutralizing the WA1/2020 strain, is much higher at 25.37 μg/mL, indicating that p11D7 is not a potently neutralizing mAb by itself.…”

“…Of note, CR3022, a Class 4 non‐neutralizing mAb against SARS‐CoV‐2 (Barnes et al ., 2020 ), derived from the original SARS‐CoV outbreak (ter Meulen et al ., 2006 ) did not recognize the Omicron RBD. This is of particular interest because we validated in this study that p11D7 has an overlapping epitope with CR3022 (Jugler et al ., 2022 ), strongly suggesting that p11D7 may itself be a Class 4 RBD‐binding mAb. However, even with an overlapping epitope, the undetermined epitope of p11D7 appears to be more conserved on the RBD than that of CR3022, due to retained binding with accumulating RBD mutations.…”

Production of two SARS-CoV-2 neutralizing antibodies with different potencies in Nicotiana benthamiana

“…A table reporting neutralization potencies of nAbs obtained in plants and mammalian cells is presented ( Table 1 ). Differences in the IC values of the same antibody obtained either in plants or mammalian cells are evident for nAbs CA1 and CB6 (Jugler et al, 2022 ). In some cases, it is difficult to directly compare data between the publications due to differences in the IC calculations, but it is evident that mAbJ08-MUT produced both in plants and mammalian cells is the only nAb endowed with potencies in the 10 ng/mL range.…”