Engineering the interactions between a plant‐produced HIV antibody and human Fc receptors

Abstract

Plants can provide a cost-effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential for precise engineering of glycosylation. Glycan structures in the antibody Fc region influence binding properties to Fc receptors, which opens opportunities for modulation of antibody effector functions. To test the impact of glycosylation in detail, on binding to human Fc receptors, different glycovariants of VRC01, a broadly neutralizing HIV monoclonal antibody, were generated in Nicotiana benthamiana and characterized. These include glycovariants lacking plant characteristic a1,3-fucose and b1,2xylose residues and glycans extended with terminal b1,4-galactose. Surface plasmon resonance-based assays were established for kinetic/affinity evaluation of antibody-FccR interactions, and revealed that antibodies with typical plant glycosylation have a limited capacity to engage FccRI, FccRIIa, FccRIIb and FccRIIIa; however, the binding characteristics can be restored and even improved with targeted glycoengineering. All plant-made glycovariants had a slightly reduced affinity to the neonatal Fc receptor (FcRn) compared with HEK cellderived antibody. However, this was independent of plant glycosylation, but related to the oxidation status of two methionine residues in the Fc region. This points towards a need for process optimization to control oxidation levels and improve the quality of plant-produced antibodies.

Characterisation of a Highly Potent and Near Pan-Neutralising Anti-HIV Monoclonal Antibody Expressed in Tobacco Plants

“…In an SPR assay measuring binding of soluble FcγRIIIa to N6 on the solid phase, there was almost one log difference in a nity between pN6 (K D 6.439 × 10 − 8 M) and mN6 (K D 8.577 × 10 − 7 M). As observed elsewhere, the difference in a nity was largely due to a difference in the dissociation constant K d (31).…”

“…We and others have shown that IgG mAbs bearing N-glycan fucosylation at either the α-1,6 position (in mammals) or the α-1,3 (in plants) have reduced binding a nity to the low a nity FcγRIIIa (CD16a) receptor(31) , (56)(57)(58)(59) which is found on natural killer cells, neutrophils and monocytes. Expressing an IgG mAb in the ΔXF N. benthamiana line was shown to restore the interaction with FcγRIIIa receptor (31). Antibody binding to FcγRIIIa is associated with antibody dependent cellular cytotoxicity (ADCC), an effector mechanism that enhances killing of virally infected cells by these immune cells (58).…”

“…In this study, the feasibility of producing bNAb N6 in plants was investigated. A glyco-engineered line of Nicotiana benthamiana (ΔXF)(30) was used to overcome potential issues with effector function and blood clearance activity (31). The puri ed protein was characterised and compared to the same antibody produced by a conventional mammalian cell expression system, in terms of antigen binding, binding kinetics and breadth of viral neutralisation, as well as glycosylation and FcγRIIIa and ADCC activity.…”

Plant-based production of highly potent anti-HIV antibodies with engineered posttranslational modifications

“…The efficacy of plant-produced versions of some of these antibodies has been tested in animal trials 7 . A remarkable achievement is the generation of Abs with engineered Fc glycosylation resulting in similar or increased Fc receptor binding activities compared to the non-glycoengineered mammalian cell-derived variants [15][16][17] .…”

Will plant-made biopharmaceuticals play a role in the fight against COVID-19?

“…Although other antibodies have shown better performance, this plant-made antibody cocktail has revealed the potential of the technology in the fight against emerging diseases [15]. Today glycoengineering approaches are available to yield specific antibody glycoforms, which will allow optimizing the functional activity and safety of the target antibodies [16]. Moreover, functional single chain antibodies are also produced in plant systems; providing simpler molecules for viral neutralization [17].…”