Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, has caused more than 4.5 million deaths worldwide. Severe and fatal cases of COVID-19 are often associated with increased proinflammatory cytokine levels including interleukin 6 (IL-6) and acute respiratory distress syndrome. In this study, we explored the feasibility of using plants to produce an anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) and examined its utility in reducing IL-6 signaling in an in vitro model, which simulates IL-6 induction during SARS-CoV-2 infection. The anti-IL6R mAb (IL6RmAb) was quickly expressed and correctly assembled in Nicotiana benthamiana leaves. Plant-produced IL6RmAb (pIL6RmAb) could be enriched to homogeneity by a simple purification scheme. Furthermore, pIL6RmAb was shown to effectively inhibit IL-6 signaling in a cell-based model system. Notably, pIL6RmAb also suppressed IL-6 signaling that was induced by the exposure of human peripheral blood mononuclear cells to the spike protein of SARS-CoV-2. This is the first report of a plant-made anti-IL-6R mAb and its activity against SARS-CoV-2-related cytokine signaling. This study demonstrates the capacity of plants for producing functionally active mAbs that block cytokine signaling and implies their potential efficacy to curb cytokine storm in COVID-19 patients.

The development of monoclonal antibodies (mAbs) has provided vast opportunities to treat a wide range of diseases from cancer to viral infections. While plant-based production of mAbs has effectively lowered the upstream cost of mAb production compared to mammalian cell cultures, further optimization of downstream processing, especially in extending the longevity of Protein A resin by an effective bulk separation step, will further reduce the overall prohibitive cost of mAb production. In this study, we explored the feasibility of using aqueous two-phase separation (ATPS) in capturing and separating plant-made mAbs from host proteins. Our results demonstrated that an anti-West Nile virus mAb (E16) was efficiently separated from most plant host proteins by a single ATPS step, comprising the mixing of plant extracts containing Hydrophobin-Protein A fusion protein (HPA) and E16 and the subsequent incubation with an inexpensive detergent. This simple ATPS step yielded a highly enriched E16 mAb preparation with a recovery rate comparable to that of Protein A chromatography. The ATPS-enriched E16 retained its structural integrity and was fully functional in binding its target antigen. Notably, HPA-based ATPS was also effective in enriching E16 from plant host proteins when both HPA and E16 were produced in the same leaves, supporting the potential of further streamlining the downstream purification process. Thus, ATPS based on plant-produced HPA in unpurified extract is a cost-effective yet efficient initial capture step for purifying plant-made mAbs, which may significantly impact the approach of mAb purification.

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.

Monoclonal antibodies (mAbs) are important proteins used in many life science applications, from diagnostics to therapeutics. High demand for mAbs for different applications urges the development of rapid and reliable recombinant production platforms. Plants provide a quick and inexpensive system for producing recombinant mAbs. Moreover, when paired with an established platform for mAb discovery, plants can easily be tailored to produce mAbs of different isotypes against the same target. Here, we demonstrate that a hybridoma-generated mouse mAb against chitinase 1 (CTS1), an antigen from Coccidioides spp., can be biologically engineered for use with serologic diagnostic test kits for coccidioidomycosis (Valley Fever) using plant expression. The original mouse IgG was modified and recombinantly produced in glycoengineered Nicotiana benthamiana plants via transient expression as IgG and IgM isotypes with human kappa, gamma, and mu constant regions. The two mAb isotypes produced in plants were shown to maintain target antigen recognition to CTS1 using similar reagents as the Food and Drug Administration (FDA)-approved Valley Fever diagnostic kits. As none of the currently approved kits provide antibody dilution controls, humanization of antibodies that bind to CTS1, a major component of the diagnostic antigen preparation, may provide a solution to the lack of consistently reactive antibody controls for Valley Fever diagnosis. Furthermore, our work provides a foundation for reproducible and consistent production of recombinant mAbs engineered to have a specific isotype for use in diagnostic assays.

Summary 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.