Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead the generation products with low efficacy. Here we report in vivo protein plants, have been shown be well suited for efficient complex glycoproteins. This was achieved by introduction an entire biosynthetic pathway Nicotiana...

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, specific immunological relevance individual mAb-associated N-glycan structures currently largely unknown, because heterogeneous profiles mAbs when produced in mammalian cells. Here we report on generation a plant-based expression platform allowing efficient production with homogeneous β1,4-galactosylated structure, major species present serum IgG. This was...

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with customized N-glycosylation pattern. Here, we demonstrate the modulation of plant glycosylation pathway toward formation human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific (i.e. β1,2-xylose core α1,3-fucose) was used transiently express human erythropoietin (hEPO) transferrin (hTF) together modified versions...

Significance Sialic acid (Sia) residues are essential monosaccharides in mammals and confer multiple biological functions. Their precise generation is important for both structure–function studies biotechnological applications. We describe a unique technology that enables the controlled of protein sialylation Nicotiana benthamiana . The plant engineering approach relies on combination endogenous glycan deconstruction introduction human capabilities. An arrangement transgenic transient...

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared non-sialylated forms. This applies particularly recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted vivo protein sialylation at great homogeneity. However the synthesis multi-sialylated N-glycans is so far restricted mammalian cells. Here we used a plant based system accomplish multi-antennary...

An accurate physical map of the location 5S and 18S-5.8S-25S rRNA genes a repetitive DNA sequence has been produced on Aegilops umbellulata Zhuk., (2n = 2x 14) chromosomes by in situ hybridization. Chromosome morphology together with hybridization pattern pSc119.2, from rye, allowed identification discrimination different chromosomes; pSc119.2 hybridizes all Ae. at telomeres, except for short arm chromosome 6U, shows intercalary sites long arms 6U 7U. The 18S-25S rDNA have mapped physically...

Previous studies have reported that plants contain negligible amounts of free or protein-bound N-acetylneuraminic acid (Neu5Ac). This is a major disadvantage for the use as biopharmaceutical expression system, since N-glycans with terminal Neu5Ac residues are important biological activities and half-lives recombinant therapeutic glycoproteins in humans. For synthesis Neu5Ac-containing N-glycans, to acquire ability synthesize its nucleotide-activated derivative, cytidine...

Fc-glycosylation of monoclonal antibodies (mAbs) has profound implications on the Fc-mediated effector functions. Alteration this glycosylation may affect efficiency an antibody. However, difficulties in production mAbs with homogeneous N-glycosylation profiles sufficient amounts hamper investigations potential biological impact different glycan residues.Here we set out to evaluate a transient plant viral based system for rapid generation glycoforms Ebola virus mAb h-13F6 was generated using...

Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued human protease inhibitor α1-antitrypsin (A1AT) Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels A1AT were expressed leaves (up to 6 mg g(-1)...

We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in Fab domain addition to conserved Fc glycosylation, as reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms CxMab differing core fucosylation (α1,3- and α1,6-linkage) were generated plant-based expression platform. These GnGn, GnGnF3, GnGnF6 variants subjected vivo further toward sialylation diversification (bisected branching structures). Mass...

Summary N‐glycosylation is critical for recombinant glycoprotein production as it influences the heterogeneity of products and affects their biological function. In most eukaryotes, oligosaccharyltransferase central‐protein complex facilitating proteins in lumen endoplasmic reticulum ( ER ). Not all potential sites are recognized vivo site occupancy can vary different expression systems, resulting underglycosylation glycoproteins. To overcome this limitation plants, we expressed Lm STT 3D, a...

Plants are attractive hosts for the production of recombinant glycoproteins therapeutic use. Recent advances in glyco-engineering facilitate elimination nonmammalian-type glycosylation and introduction missing pathways customized N-glycan formation. However, some therapeutically relevant exhibit unwanted truncated (paucimannosidic) N-glycans that lack GlcNAc residues at nonreducing terminal end. These paucimannosidic increase product heterogeneity may affect biological function drugs. Here,...

Human angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus (SARS-CoV-2) binding and entry. Administration of high concentrations soluble ACE2 can be utilized as a decoy to block interaction virus with cellular receptors potentially used strategy treatment or prevention disease 2019. heavily glycosylated its glycans impact on SARS-CoV-2 spike protein infectivity. Here, we describe production recombinant ACE2-fragment...

Abstract Intracellular trafficking and subcellular deposition are critical factors influencing the accumulation posttranslational modifications of proteins. In seeds, these processes not yet fully understood. this study, we set out to investigate intracellular transport, final destination, N-glycosylation status, stability fusion recombinant single-chain variable fragments crystallizing fragment an antibody (scFv-Fc) two antiviral monoclonal antibodies (2G12 HA78). The scFv-Fcs were...

Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production rhEPO currently restricted to mammalian cell expression systems because rhEPO's highly complex glycosylation pattern, which major determinant for drug-efficacy. Here we evaluate ability plants produce different glycoforms rhEPO. cDNA constructs were delivered Nicotiana benthamiana (N. benthamiana) and transiently expressed by viral based system. Expression levels up 85...

Human butyrylcholinesterase (BChE) is considered a candidate bioscavenger of nerve agents for use in pre- and post-exposure treatment. However, the presence functional necessity complex N-glycans (i.e. sialylated structures) challenging issue respect to its recombinant expression. Here we transiently co-expressed BChE cDNA model plant Nicotiana benthamiana with vectors carrying genes necessary planta protein sialylation. Site-specific sugar profiling secreted (rBChE) collected from...

A pioneering strategy to expedite drug discovery for PD-1/PD-L1 axis inhibition is offered by an integrated approach combining computer-aided design (CADD) tools, QSAR modelling, repurposing, and molecular docking.

Abstract Key message Plant-made PD1–Fc fusions engineered for optimized glycosylation and Fc-receptor engagement are highly efficient in blocking PD1/PDL1 interactions can be cost-effective alternatives to antibody-based immune checkpoint inhibitors. Immune inhibitors (ICIs) antibodies receptors that have pivotal roles during T-cell activation processes. The programmed cell death 1 (PD1) regarded as the primary targeting PD1 or its ligand PDL1 revolutionized immunotherapy of cancer. However,...

Newly emerging SARS-CoV-2 variants of concern (VOCs) continue to drive COVID-19 waves and are typically associated with immune escape increased resistance current therapeutics including monoclonal antibodies. By contrast, VOCs still display strong binding the host cell receptor ACE2. Consistent these properties, we have now found that a soluble ACE2-Fc decoy produced in glycoengineered plants effectively neutralizes different isolates exhibits even potency against as compared an ancestral...

We have investigated the organization of transgenes delivered by particle bombardment into wheat genome, combining conventional molecular analysis with fluorescence in situ hybridization (FISH) and three-dimensional confocal microscopy. selected a representative population transformed lines carried out expression analysis. FISH on metaphase chromosomes showed that transgene integration sites were often separated considerable lengths genomic DNA (>1 Mbp), or could even be opposite chromosome...

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little known about overall impact non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version β1,4-galactosyltransferase ((ST)GalT). While some grew normally, other...

Summary Plants can provide a cost‐effective and scalable technology for production of therapeutic monoclonal antibodies, with the potential precise engineering glycosylation. Glycan structures in antibody Fc region influence binding properties to receptors, which opens opportunities modulation effector functions. To test impact glycosylation detail, on human different glycovariants VRC 01, broadly neutralizing HIV antibody, were generated Nicotiana benthamiana characterized. These include...