Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana

N-glycan engineering has dramatically evolved for the development and quality control of recombinant antibodies. Fc region of IgG contains two N-glycans whose galactose terminals on Fc-glycan have been shown to increase the stability of CH2 domain and improve effector functions. Nicotiana benthamiana has become one of the most attractive production systems for therapeutic antibodies. In this study, Varlilumab, a CD27-targeting monoclonal antibody, was transiently produced in fresh leaves of soil-grown and hydroponic-grown N. benthamiana, resulted in the yield of 174 and 618 µg/gram, respectively. However, the IgG produced in wild-type N. benthamiana lacked the terminal galactose residues in its N-glycan. Therefore, N-glycan engineering was applied to fine-tune recombinant antibodies produced in plant platforms. We further co-expressed IgG together with murine β1,4-galactosyltransferase (β1,4-GALT) to modify plant N-glycan with β1,4-linked Gal residue(s) and Arabidopsis thaliana β1,3-galactosylatransferase (β1,3-GALT) to improve galactosylation. The co-expression of IgG with each of GALTs successfully resulted in modification of N-glycan structures on the plant-produced IgG. Notably, IgG co-expressed with murine β1,4-GALT in soil-grown N. benthamiana had 42.5% of N-glycans variants having galactose (Gal) residues at the non-reducing terminus and 55.3% of that in hydroponic-grown N. benthamiana plants. Concomitantly, N-glycan profile analysis of IgG co-expressed with β1,3-GALT demonstrated that there was an increased efficiency of galactosylation and an enhancement in the formation of Lewis a structure in plant-derived antibodies. Taken together, our findings show that the first plant-derived Varlilumab was successfully produced with biantennary β1,4-galactosylated N-glycan structures.Nguyen K.D., Kajiura H., Kamiya R., et al. Production and N-glycan engineering of Varlilumab in Nicotiana benthamiana. Frontiers in Plant Science 14, 1215580 (2023); https://doi.org/10.3389/fpls.2023.1215580

Structure–function studies of ultrahigh molecular weight isoprenes provide key insights into their biosynthesis

Some plant trans-1,4-prenyltransferases (TPTs) produce ultrahigh molecular weight trans-1,4-polyisoprene (TPI) with a molecular weight of over 1.0 million. Although plant-derived TPI has been utilized in various industries, its biosynthesis and physiological function(s) are unclear. Here, we identified three novel Eucommia ulmoides TPT isoforms—EuTPT1, 3, and 5, which synthesized TPI in vitro without other components. Crystal structure analysis of EuTPT3 revealed a dimeric architecture with a central hydrophobic tunnel. Mutation of Cys94 and Ala95 on the central hydrophobic tunnel no longer synthesizd TPI, indicating that Cys94 and Ala95 were essential for forming the dimeric architecture of ultralong-chain TPTs and TPI biosynthesis. A spatiotemporal analysis of the physiological function of TPI in E. ulmoides suggested that it is involved in seed development and maturation. Thus, our analysis provides functional and mechanistic insights into TPI biosynthesis and uncovers biological roles of TPI in plants

Engineering the Plant Factory for the Production of Biologics and Small-Molecule Medicines

Plant gene transfer achieved in the early ‘80s paved the way for the exploitation of the potential of gene engineering to add novel agronomic traits and/or to design plants as factories for high added value molecules. For this latter area of research, the term "Molecular Farming" was coined in reference to agricultural applications in that major crops like maize and tobacco were originally used basically for pharma applications. The concept of the “green biofactory” implies different advantages over the typical cell factories based on animal cell or microbial cultures already when considering the investment and managing costs of fermenters. Although yield, stability, and quality of the molecules may vary among different heterologous systems and plants are competitive on a case-to-case basis, still the “plant factory” attracts scientists and technologists for the challenging features of low production cost, product safety and easy scale up. Once engineered, a plant is among the cheapest and easiest eukaryotic system to be bred with simple know-how, using nutrients, water and light. Molecules that are currently being produced in plants vary from industrial and pharmaceutical proteins, including medical diagnostics proteins and vaccine antigens, to nutritional supplements such as vitamins, carbohydrates and biopolymers. Convergence among disciplines as distant as plant physiology and pharmacology and, more recently, as omic sciences, bioinformatics and nanotechnology, increases the options of research on the plant cell factory. “Farming for Pharming” biologics and small-molecule medicines is a challenging area of plant biotechnology that may break the limits of current standard production technologies. The recent success on Ebola fighting with plant-made antibodies put a spotlight on the enormous potential of next generation herbal medicines made especially in the name of the guiding principle of reduction of costs, hence reduction of disparities of health rights and as a tool to guarantee adequate health protection in developing countries.Plant gene transfer achieved in the early ‘80s paved the way for the exploitation of the potential of gene engineering to add novel agronomic traits and/or to design plants as factories for high added value molecules. For this latter area of research, the term "Molecular Farming" was coined in reference to agricultural applications in that major crops like maize and tobacco were originally used basically for pharma applications. The concept of the “green biofactory” implies different advantages over the typical cell factories based on animal cell or microbial cultures already when considering the investment and managing costs of fermenters. Although yield, stability, and quality of the molecules may vary among different heterologous systems and plants are competitive on a case-to-case basis, still the “plant factory” attracts scientists and technologists for the challenging features of low production cost, product safety and easy scale up. Once engineered, a plant is among the cheapest and easiest eukaryotic system to be bred with simple know-how, using nutrients, water and light. Molecules that are currently being produced in plants vary from industrial and pharmaceutical proteins, including medical diagnostics proteins and vaccine antigens, to nutritional supplements such as vitamins, carbohydrates and biopolymers. Convergence among disciplines as distant as plant physiology and pharmacology and, more recently, as omic sciences, bioinformatics and nanotechnology, increases the options of research on the plant cell factory. “Farming for Pharming” biologics and small-molecule medicines is a challenging area of plant biotechnology that may break the limits of current standard production technologies. The recent success on Ebola fighting with plant-made antibodies put a spotlight on the enormous potential of next generation herbal medicines made especially in the name of the guiding principle of reduction of costs, hence reduction of disparities of health rights and as a tool to guarantee adequate health protection in developing countries