Golgi engineering of CHO cells by targeted integration of glycosyltransferases leads to the expression of novel Asn-linked oligosaccharide structures at secretory glycoproteins
© Reinl et al.; licensee BioMed Central Ltd. 2013
Published: 4 December 2013
Background and novelty
N-glycans constitute an important information carrier in protein-driven signaling networks. Amongst others, N-glycans contribute to protein folding quality, adjust protein turnover and operate as address label for targeting proteins to specific cells and tissues . Hence, the composition of N-glycans attached to recombinant glycoprotein therapeutics is vital for in-vivo therapeutic efficacy and strongly depends on the choice of the expression host [2, 3]. Due to absence or silencing of glycosyltransferase genes homologue to human enzymes, biotechnologically used cell lines are limited by their intrinsic glycosylation machinery and produce host specific glycoforms.
The design of new quality-optimized and functionally improved biopharmaceuticals with properties conferred by host cell unrelated N-glycans requires a rational Golgi engineering strategy. Here, we apply GET, a system that enables the positioning of a desired catalytic glycosyltransferase activity into a favorable localization within the intracellular glycosylation machinery, to suspension CHO cells developed to secrete suitable amounts (200 μg/ml) of Cetuximab as a model glycoprotein. The presented Golgi engineering project aims in the extension of the intrinsic glycosylation repertoire enabling CHO cells to produce new human-type glycosylation motifs as indicated in Figure 1A: (i) GalNAcβ1,4GlcNAc-R (LacdiNAc, LDN),(ii) GlcNAc in β1,4 linkage to central mannose residue (bisecting GlcNAc, bGN), (iii) Galβ1,4(Fucα1,3)GlcNAc-R (LewisX, LeX) and (iv) NeuAcα2,3Galβ1,4 (Fucα1,3)GlcNAc-R (Sialyl-LewisX, sLeX). To assemble (ii) and (iv), we co-express GnT3 and FT7. As shown earlier, the latter enzyme catalyzes fucosylation exclusively of (iv). Therefore, we included in our study a variant of FT6 that is targeted to the early Golgi compartment with the aim to additionally generate structure (iii) [6, 7]. The uncommon LDN motif (i) which is e.g. detected on lutropin is assembled by human B4GalNT3 [8, 9]. We analyze oligosaccharides released from the products of genetically engineered CHO cells based on the resolution of single glycosylation sites of VH- and CH2- glycopeptides by quantitative NP-HPLC-FLD and use our comprehensive oligosaccharide standard library to identify novel oligosaccharide motifs.
Cloning of human glycosyltransferases and engineering of VARFT6  as well as construction of pGET expression plasmids encoding either the heavy and light chain of Cetuximab or the glycosyltransferase cDNAs was done acc. to standard DNA technologies. A stable clone with Cetuximab titers of 200 μg/ml and doubling times of 25 hours was selected after transfection of pGET-Cetuximab in CHO cells. This clone was either mock- or co-transfected with pGET plasmids encoding the indicated glycosyltransferases. After shake flask subcultivation for 72 h Cetuximab was purified from supernatants, digested and applied to RP-HPLC peptide mapping. CH2- and VH-domain glycopeptides were separated and oligosaccharides were enzymatically released. After 2-AB labeling, the isolated oligosaccharides were subjected to quantitative NP-HPLC-FLD and ESI-TOF-MS and MS/MS analysis. Oligosaccharide structures were unambiguously identified in comparison to GlycoThera's reference standard oligosaccharide library.
Results and discussion
The heterologous co-expression of wildtype B4GalNT3, GnT3 and FT7 and genetically modified FT6 results in the formation of the uncommon LacdiNAc motif (ca. 40%), the LewisX and di-LewisX structures (ca. 50%) and Sialyl-LewisX (ca. 15%) almost exclusively on oligosaccharides from the VH-domain. Relevant modification of both VH-domain (ca. 40%) and CH2-domain glycans (ca. 30%) is only achieved by GnT3 catalyzed attachment of bisecting GlcNAc. In addition, glycosyltransferase co-expression leads to charge state reduction of oligosaccharides by depletion of suitable acceptors for endogenous sialyltransferases. The strongest reduction in the content of neuraminic acid at VH-domain was observed by co-expression of VARFT6 (ca. 55% reduction) and WTB4GalNT3 (ca. 50% reduction).
As a conclusion, Golgi engineering endows CHO cells to assemble significant amounts of LacdiNAc, bisecting GlcNAc, LewisX and Sialyl-LewisX to Cetuximab N-glycans (Figure 1B and Figure 2). Therefore, our glycosylation engineering strategy provides a tool to produce tailored N-glycosylation variants with defined structural motifs. As demonstrated, the tailored addition of bisecting GlcNAc to CH2-domain N-glycans increases ADCC of an αCD20 therapeutic mAB . We therefore assume that the presented structural motifs exhibit novel therapeutic properties (ADCC, CDC, tissue specificity, serum half-life). Our strategy represents a relevant basis for the development of biotherapeutics and biobetters with potentially improved pharmacokinetics, pharmacodynamics, safety properties and in vivo therapeutic efficacy.
- Varki A, Lowe JB: Biological Roles of Glycans. Essentials of Glycobiology. Edited by: Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME. 2009, Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press, Chapter 6, 2Google Scholar
- Sinclair AM, Elliott S: Glycoengineering: the effect of glycosylation on the properties of therapeutic proteins. J Pharm Sci. 2005, 94: 1626-1635.View ArticlePubMedGoogle Scholar
- Grabenhorst E, Schlenke P, Pohl S, Nimtz M, Conradt HS: Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells. Glycoconj J. 1999, 16: 81-97.View ArticlePubMedGoogle Scholar
- Erbitux® (Cetuximab): Prescribing Information. Bristol-Myers Squibb (1236886B3, Rev. March 2013)Google Scholar
- Commins SP, Platts-Mills TAE: Allergenicity of Carbohydrates and Their Role in Anaphylactic Events. Curr Allergy Asthma Rep. 2010, 10: 29-33.PubMed CentralView ArticlePubMedGoogle Scholar
- Grabenhorst E, Nimtz M, Costa J, Conradt HS: In Vivo Specificity of Human α1,3/4-Fucosyltransferases III-VII in the Biosynthesis of LewisX and Sialyl LewisX Motifs on Complex-type N-Glycans. J Biol Chem. 1998, 273: 30985-30994.View ArticlePubMedGoogle Scholar
- Grabenhorst E, Conradt HS: The cytoplasmic, transmembrane, and stem regions of glycosyltransferases specify their in vivo functional sublocalization and stability in the Golgi. J Biol Chem. 1999, 274: 36107-36116.View ArticlePubMedGoogle Scholar
- Sato T, Gotoh M, Kiyohara K, Kameyama A, Kubota T, Kikuchi N, Ishizuka Y, Iwasaki H, Togayachi A, Kudo T, Ohkura T, Nakanishi H, Narimatsu H: Molecular cloning and characterization of a novel human beta 1,4-N-acetylgalactosaminyltransferase, beta 4GalNAc-T3, responsible for the synthesis of N,N'-diacetyllactosediamine, galNAc beta 1-4GlcNAc. J Biol Chem. 2003, 278: 47534-47544.View ArticlePubMedGoogle Scholar
- Fiete D, Srivastava V, Hindsgaul O, Baenziger JU: A hepatic reticuloendothelial cell receptor specific for SO4-4GalNAc beta 1,4GlcNAc beta 1,2Man alpha that mediates rapid clearance of lutropin. Cell. 1991, 67: 1103-1110.View ArticlePubMedGoogle Scholar
- Davies J, Jiang L, Pan LZ, LaBarre MJ, Anderson D, Reff M: Expression of GnTIII in a recombinant anti-CD20 CHO production cell line: Expression of antibodies with altered glycoforms leads to an increase in ADCC through higher affinity for FC gamma RIII. Biotechnol Bioeng. 2001, 74: 288-294.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.