Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies

Abstract

N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies.

In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants

“…Rituximab purification was carried out as described earlier [45]. Antibody samples were analyzed on a 4–12% Bis-Tris gradient NuPAGE™ gel under reducing conditions according to the manufacturer’s protocol (Life Technologies, Carlsbad, CA, USA).…”

“…The increased ADCC activity was verified using effector cells carrying both FcγRIIIa-V158 and FcγRIIIa-F158 allotypes. Several strategies could be implemented to modulate the plant-specific glycans: (i) Protein containment in the Endoplasmic Reticulum (ER) using specific signal sequences (i.e., SEKDEL sequence: Ser-Glu-Lys-Asp-Glu-Leu) [43], (ii) knockdown of fucosyltransferase and xylosyltransferase enzymes in N. benthamiana with RNA interference (RNAi) technology [18], (iii) knockout of fucosyltransferase and xylosyltransferase enzymes in N. benthamiana using gene editing [44], and (iv) replacement of plant glycans with human glycans through glyco-remodeling [45]. Gene editing using sequence-specific transcription-activator-like effector nucleases (TALENs) was only partially effective [44], while the use of transgenic knockdown lines at manufacturing require more exigent containment and cleaning procedures.…”

From Farm to Finger Prick—A Perspective on How Plants Can Help in the Fight Against COVID-19

“…Antigens are also decorated with sugars in a process known as glycosylation and these sugars can impact their binding and detection of antibodies that indicated prior exposure to SARS-CoV-2. Fortunately, plant glycosylation can be altered as needed through both process engineering approaches (Castilho et al, 2010 ; Kommineni et al, 2019 ) and/or post processing modifications (Bennett et al, 2018 ), although the latter would add some additional cost.…”

Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor

“…Several strategies exist to modify a glycoprotein’s N -glycan structures in planta, such as glycoengineering of the host cells using CRISPR/Cas9 genome editing to knock out β(1,2)-xylosyltransferase ( XylT ) genes and α(1,3)-fucosyltransferase ( FucT ) genes [ 10 , 11 , 12 ] and RNA interference (RNAi) technology to downregulate XylT and FucT genes [ 13 , 14 , 15 , 16 , 17 ], targeting of the protein to specific organelles [ 18 , 19 , 20 , 21 ], addition of compounds to alter the function of glycan-modifying enzymes [ 4 , 22 , 23 , 24 , 25 , 26 ], and in vitro glycan remodeling using chemoenzymatic reactions [ 27 ]. In this work, we utilize kifunensine, a potent and highly specific inhibitor of α-mannosidase I in both plant and animal cells resulting in production of glycoproteins containing predominantly Man 8 GlcNAc 2 (Man8) and Man 9 GlcNAc 2 (Man9) structures [ 28 ], to a rice cell suspension culture grown in a bioreactor to inhibit α-mannosidase I activity.…”