17.09.20151 Plants as Expression System for Recombinant Therapeutic Glycoproteins University of...

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  • **Plants as Expression System for Recombinant Therapeutic GlycoproteinsUniversity of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biologywww.boku.ac.atHerta Steinkellner, Richard Strasser and Josef GlsslINTERNATIONAL SCIENTIFIC CONFERENCEEARTH BIORESOURCES AND ENVIRONMENTAL BIOSAFETY: CHALLENGES AND OPPORTUNITIESKyiv, Ukraine, November 4-7, 2013

  • Glycosylation of proteinsN-Glycosylation: Asn-linked glycosylation (Asn-X-Ser/Thr) N-glycansO-Glycosylation: Ser/Thr-linked glycosylation O-glycansP. Stanley, 2011 Cold Spring Harbor Laboratory PressGlycosphingolipids

  • N-glycosylationFunction of N-glycans: - Protein folding and stability- Protein targeting (e.g. for lysosomal enzymes) - Protein-protein or protein-carbohydrate interactions (lectins) - Biological activity of proteins (e.g. effector functions of IgGs)- Control of protein half-life (e.g. erythropoietin)

  • *Current expression systems generate a mixture of glycoformsSubstantial deficits exist in understanding the role of specific glycosylation patterns on therapeutic proteins like monoclonal antibodiesWell defined glycoforms on recombinant proteins are urgently neededExpression systems are needed which produce tailor-made N-glycan structuresWhy glyco-engineering ?Changes in N-glycan structure may significantly affect protein confirmation and pharmacokinetic behaviour, thus influencing e.g. antigen binding and effector functions in case of monoclonal antibodies

  • *General goals:

    Production of recombinant glycoproteins with a defined, homogeneous glycosylation profile in order to study

    the impact of glycosylationthe therapeutic potency of various glycoforms

    Further development of plant expression systems for therapeutically relevant glycoproteins:

    Why glyco-engineering ?

  • *

    Plant expression systems are rapidly evolving as expression systems for therapeutically relevant proteins, since they areconvenient, biologically safe cost-effective

    However, since plants differ in certain aspects of their N-glycosylation pathway from that in human, humanization of the N-glycan biosynthetic pathway is needed in order to avoid immunological problemsget authentic N-glycan structures

    Why glyco-engineering in plants?

  • N-glycosylation in mammalian cellsProminent example: monoclonal antibodies (mAbs):IgG1 heavy chain has conserved N-glycosylation site (Asn 297) N-glycan structure influences biological activity of antibodies

  • Schematic presentation of the N-glycosylation pathways in humans, yeast, insect cells and plantsThe common ER-resident oligosaccharide precursor acts as starting point for further modifications along the Golgi apparatusLegend:

  • *cis Golgimedial Golgitrans GolgiN-Glycan Processing in Plants vs. Mammalsb1,4-GalT1,6-FucTb1,2-XylTMammalsPlant and mammalian N-glycan processing steps differ in the late Golgi stepsN-acetylglucosamineMannoseFucoseGalactoseXylosePlantsb1,3-GalT1,4-FucT1,3-FucTSialic acid2,6-SiaT

  • Engineering of the N-Glycan Processing Pathway in PlantsRemoval of 1,2-xylosyltransferase core 1,3-fucosyltransferase

    Transformation with 1,4-galactosyltransferaseby knock out (A. thaliana) RNAi (N. benthamiana)Engineering of the sialic acid pathway by:

    Transformation of six genes required for the biosynthesis of CMP-Neu5Ac and transport into the Golgi lumen

    Transformation with 2,6-sialyltransferaseStrategy:Goal: Humanised N-glycan structuresPlant-type N-glycanHumanized N-glycan

  • Reconstruction of the human sialylation pathway in plantsThe genes for 6 proteins had to be introduced into plant cell, permitting the biosynthesis of sialic acid (Neu5Ac) , its activation, transport into the Golgi, and transfer onto terminal galactoseA. Castilho et al., J. Biol. Chem.285, 15923, 2010

  • Different functional activities of monoclonal antibody (mAb) N-glycoforms2 Examples of mAbs against viruses:anti HIV antibdy 2G12:(Polymun, Dept. Biotechnology, BOKU, Vienna)anti EBOLA virus antibody 13F6(Mapp Biopharmaceutical, CA, USA)

  • mAb 2G12 binding to Fc receptor IIIa (CD16a) Functional activities of mAbs glycoformsFucose deficient mAb 2G12 exhibits higher binding to CD16

    Higher anti HIV activity of mAb 2G12D.N. Forthal et al., J. Immunol. 185, 6876, 2010

  • mAb 13F6 (anti Ebola virus)Virus protection assay in mice Kevin WhaleyFunctional activities of mAbs glycoforms

  • *Summary and OutlookIt is now possible to produce complex human proteins for therapeutic purposes, largely correctly folded and N-glycosylated, in plantsPlants have demonstrated a high degree of tolerance to changes in the N-glycosylation pathway, allowing recombinant proteins to be modified into human-like structures Glyco-engineering has paved the way to fully humanize the plant N-glycosylation pathwayFrequently the results are a largely homogeneously glycosylated protein, enabling to studythe impact of glycosylationthe therapeutic potency of various glycoforms There is increasing evidence for the significance of proper N-glycosylation for the efficacy of biopharmaceuticals Glyco-engineering has become an important issue not only for academia but also for the biopharmaceutical industry.

  • *AcknowledgementsDepartment of Applied Genetics and Cell BiologyHerta SteinkellnerRichard StrasserAlexandra CastilhoPia GattingerJakub Jez

    Department of Chemistry (N-glcosylation analyses)Friedrich AltmannJohannes Stadlmann

    Department of Biotechnology (HIV testing)Renate KunertHeribert Quendler

    Supported by:Austrian Science FundEuropean CommissionBOKU - University of Natural Resources and Life Sciences, Vienna

  • *09.07.2012*

    University of Natural Resources and Life Sciences, Vienna (BOKU)

    congratulates to theProf. Dr. Josef GlsslVice Rector for Research and International Research Collaboration

    [email protected] Anniversary of NULES of Ukraineand the 15th Anniversary of GCHERA

    ***Slide 4: GLYCOPROTEIN PROCESSINGGlycoprotein processing occurs in the Golgi membranes where specific enzymes involved are active. Although proteins can undergo different glycosylation they all share a common glycan structure known as core unit. Plant and mammalian N-Glycan maturation differ only in the late Golgi apparatus where complex N-Glycans are formed. In mammalians the core structure is extended by the addition of Gal 1 to 4 linkage and latter sialic acid whereas in plants the core structure is substituted by xylose and fucose in 1 to 3 and not in 1 to 6 like observed in mammals. These two epitopes are plant specific and are known to be extremely immunogenic in mammals. Another difference is although plants can have gal extended branches they are in 1 to 3 and plants complex N-glycans lack sialic acid because they lack the two enzymes to perform these two linkages beta 1-4 galactosyltransferases and alpha 2.6 sialyltransferasePLANT MOLECULAR FARMING Mammalian cells typically produced proteins with complex glycan structures terminated by the addition of Sialic Acid.The half-life and therapeutic potency of most glycoproteins (with exception for antibodies) is dependent on the presence of terminal sialic acid. Probably because of its negative charge and terminal position on many glycans, sialic acid plays a unique and significant effect on the protein: Stabilization, antigenicity, in vivo circulatory half-life and also has been implicated in process such as cell-cell signalling and cell interactionsThere are many reports about therapeutic proteins being produced in plants. Besides other important aspects, the fact that plants are able to glycosylate proteins to some extent similar to mammals makes them a more accepted host when compared to other systems like bacteria, yeast and fungi. Nevertheless, the production of recombinant therapeutic proteins in plants requires them to produce more mammalian-like proteins and for that modifications on the plant N-glycosylation pathway are needed not just to overcome the immunogenicity of plant-specific sugar residues but also to target the insertion of particular terminal sugar residues not present in plant complex N-glycans.Promising progress in engineering the plant glycosylation has already been made. The knockout of (1,3)-fucosyltransferase and the (1,2)-xylosyltransferase genes prevent the production of plant-specific glyco-epitopes without affecting protein secretion. Also, the expression of mammalian (1,4)-galactosyltransferase is gaining success on the production of plant N-glycans with terminal (1,4)-galactose residues*Mice received mAb IP 1 day prior to challenge with 1000 pfu of mouseadapted Ebola Zaire.The endpoint was survival.