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Customized protein glycosylation to improve


biopharmaceutical function and targeting
Linde Van Landuyt1,2,3, Chiara Lonigro1,2,3, Leander Meuris1,2 and
Nico Callewaert1,2

For a long time, glycoprotein production has been limited by the Introduction
inherent properties of production hosts. Glycosylation of Over the first 40 years of its existence, the biopharma-
biopharmaceuticals has been regarded as a necessary evil, ceuticals sector has been settling on a limited toolbox of
often needed for protein folding or function, but also a source of expression hosts with validated scale-up and regulatory
heterogeneity, complicating downstream processing and track record. Broadly, Escherichia coli is used for easy-to-
product characterization. This has strongly determined the fold proteins. Mammalian cell lines, in particular Chinese
choice of production hosts. Over the last few decades, Hamster Ovary (CHO) and to a lesser extent Human
numerous glycoengineering efforts have helped solving this Embryonic Kidney cells (HEK293) and some other hosts
problem. Moreover, insights from fundamental studies have are used for complex proteins. These proteins often
made it possible to improve therapeutic protein functionality require human-type glycosylation. Yeasts are used for
through careful glycoengineering. Here, we will focus on how proteins that benefit from a eukaryotic secretory system
production host and in vitro glycoengineering approaches folding environment, when glycans do not necessarily
allow to design biopharmaceuticals with glycans that impart need to be ‘human-type’. Insect cell lines have found
improved functionality. An important branch of research particular use for the production of virus-like particle
explores how glycosylation can be tuned to improve vaccines.
pharmacokinetics and reduce glycan heterogeneity of
therapeutics. Furthermore, antibody glycoengineering to Protein glycosylation is the set of post-translational
obtain homogeneous, defined glycan structures has been a modifications resulting from enzymatic addition of car-
major focus. An example of this is the production of Fc glycans bohydrates to proteins. It is the most widely occurring
without core fucose, exhibiting tremendously improved modification on proteins that pass the secretory system.
Antibody-Dependent Cell Cytotoxicity (ADCC). In the last part, The ER N-glycosylation pathway is conserved in all
glycoforms that allow for improved (subcellular) targeting and eukaryotes currently used as biopharmaceutical protein
cellular uptake, a field that opens possibilities for enzyme production hosts. It is involved in protein folding and
replacement therapies and vaccine development, will be quality control, especially for more complex proteins
highlighted. that contain disulphide bonds [1]. In the Golgi, a variety
of glycosyltransferases is responsible for cell-type-spe-
cific protein glycosylation. Given the dynamic nature of
the Golgi, resident glycosidases and glycosyltrans-
ferases often cannot modify all substrate molecules
Addresses
1
VIB-UGent Center for Medical Biotechnology, Belgium before these are transported away or before competing
2
Department of Biochemistry and Microbiology, Ghent University, transferases modify the molecule. Consequently, Golgi
Technologiepark 927, B-9052 Gent, Belgium glycosylation pathways result in a heterogeneous mix-
ture of glycan intermediates on secreted (therapeutic)
Corresponding author: Callewaert, Nico (Nico.Callewaert@ugent.vib.be)
3
These authors contributed equally.
proteins (Figure 1). Apart from being species-specific,
glycan profiles are also cell type-specific and influenced
Current Opinion in Biotechnology 2019, 60:17–28 by environmental parameters.
This review comes from a themed issue on Pharmaceutical
biotechnology Until about 2000, the protein expression community
Edited by Yvonne Y Chen and James A Van Deventer
considered these cell type-specific (and often undesired)
glycan structures and heterogeneity as a given, coping
For a complete overview see the Issue and the Editorial
with it through appropriate choice of host combined with
Available online 13th December 2018 process engineering and downstream purification opti-
https://doi.org/10.1016/j.copbio.2018.11.017 mization, often at great cost. From the late 90s onwards,
0958-1669/ã 2018 Elsevier Ltd. All rights reserved. biosynthetic engineering approaches allowed to over-
come the limitations imposed by glycosylation. Over
the past two decades, work has focused on customizing
glycan biosynthetic pathways to enhance therapeutic
protein functionality. This is a maturing research area,
with several glycoengineered products presently on the

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18 Pharmaceutical biotechnology

Figure 1

mRNA

Ribosome Cytoplasm
ER lumen cis Golgi
LLO OST
assembly

N-glycans are
important in folding
and quality control

Mammalian Golgi Insect Golgi Plant Golgi Yeast Golgi

N-Acetylglucosamine (GlcNAc) Galactose (Gal) Phosphate

Mannose (Man) N-Acetylneuraminic acid (Neu5Ac)

Fucose (Fuc) Xylose (Xyl)


Current Opinion in Biotechnology

N-glycosylation pathway across the eukaryotic expression hosts.


N-glycosylation starts in the ER with lipid linked oligosaccharide (LLO) assembly, resulting in a precursor N-glycan that is transferred most often
cotranslationally onto an unfolded protein by an oligosaccharyltransferase (OST). After this, N-glycans assist in glycoprotein folding and folding
quality control, resulting in Man8GlcNAc2-containing proteins being exported to the cis Golgi (purple framework). Man8GlcNAc2 serves as a
substrate for mannosyltransferases in the Golgi of yeasts, leading to hypermannosylated structures. In other cells, like mammalian, insect or plant
cells, the protein’s Man8GlcNAc2 are first trimmed to Man5GlcNAc2 and are eventually modified with a first b1,2-N-acetylglucosamine in the cis
Golgi (red framework). This product is the starting point for the synthesis of complex type N-glycans in mammalian cells, paucimannosidic N-
glycans in insect cells or a1,3-fucose and b1,2-xylose-containing (paucimannosidic) N-glycans in plants. In mammalian cells, lysosomal enzymes
are modified with mannose-6-phosphate-containing N-glycans, which are important in transport of such enzymes to the lysosomes.

market. This review will thus focus on how glycans can Glycoengineering for improved
be customized beyond simply mimicking natural gly- pharmacokinetics or reduced heterogeneity
cans, to obtain defined glycans that improve therapeutic Much effort is presently being invested in developing new
function. biopharmaceuticals that allow reduced or more convenient

Current Opinion in Biotechnology 2019, 60:17–28 www.sciencedirect.com


Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 19

dosing, lowering costs and increasing patient comfort. resulting in a threefold increased hydrodynamic volume
Many strategies have focused on improving bioavailability and 30-fold increased half-life [14]. This approach
and therapeutic response through increased serum half- required fusing the ScFv to an NCAM Ig5 domain
life. Traditionally, chemical conjugation to polyethylene (which contains a polysialylation site) and a fibronectin
glycol (PEGylation) has been used, mainly for small pro- 1 polysialyltransferase docking domain. Coexpression of
teins, to increase their hydrodynamic size, thus reducing the fusion construct and the human poly-a2,8-sialyl-
renal clearance [2]. Besides this, the use of newly intro- transferase in HEK293S cells resulted in polysialylation
duced N-glycosylation sites has been exploited. N-glycans of the Ig5 domain.
are much larger per mass unit as compared to folded
proteins, making them effective for increasing a Nicotiana benthamiana has also been extensively engi-
therapeutic’s size. This approach for example lead to the neered to obtain PSA in planta (Figure 2A). This required
development of longer circulating forms of EPO [3]. introducing the biosynthesis pathway for sialylated com-
plex type N-glycans into a b1,2-xylosyl-fucosyltransferase
More recently, glycosyltransferases have enabled site- and core a1,3-fucosyltransferase knockout plant
specific PEG conjugation to glycans (GlycoPEGylation) (DXTFT) first. Transient expression of human sialyl-
[4]. O-glycans of several growth factors (G-CSF, IFN- transferases ST8SiaII and ST8SiaIV then allowed for in
a2b, GM-CSF) and coagulation factors (Factor VIIa, VIII vivo synthesis of PSA with more than 40 units, without
and IX) have been modified with PEGylated N-acetyl- impacting protein yield and plant growth [15–17,18].
neuraminic acid (NANA), using a-N-acetylgalactosami- However, in vivo polysialylation in a eukaryotic host
nide a2,6-sialyltransferase 1 (ST6GALNAC1) and a always requires fusion of the therapeutic protein to one
PEG-modified CMP-NANA. Methods for (glyco)PEGy- of the naturally occurring polysialylated protein domains
lation have been reviewed recently [5]. (e.g. NCAM, Synaptic cell adhesion molecule, neuropilin
2) and only certain cell types can accomplish it. An
However, even if proteins are sufficiently large to escape engineered E. coli strain (Figure 2b) allowed circumvent-
renal clearance, they can be readily cleared by receptors ing this problem: recombinant GFP with up to 50% of
on macrophages (mannose receptor) and hepatocytes GlcGalSia2–10 glycans and designed ankyrin repeat pro-
(asialoglycoprotein receptor) when their glycans are teins with up to 28-fold increased circulation times have
incompletely sialylated. Extending biopharmaceuticals’ been generated [19]. Although promising, the strains
circulatory half-life by sialyltransferase coexpression in suffered from reduced bacterial growth and low protein
various production hosts emerged soon after sequencing yield. Further strain engineering may solve this.
of the first sialyltransferases [6–8].
Several conceptually new in vivo approaches geared
Like PEGylation, poly-a2,8-sialylation (PSA) also towards reducing N-glycan and O-glycan (micro)hetero-
increases a therapeutic’s hydrodynamic radius, but with geneity have also been introduced during the last decade.
the added advantage of not being foreign for the human GlycoDelete technology offers a strategy to reduce N-
immune system. In a first approach, PSA of prokaryotic glycan heterogeneity. Starting from GnTI / HEK293 or
origin was conjugated to lysine e-aminogroups of thera- Arabidopsis thaliana, which produce high-mannose type
peutics. This was accomplished by PSA oxidation at the glycans, a Golgi-targeted ENGase, EndoT, allows for in
non-reducing end, followed by reductive amination- vivo removal of all N-glycans. The N-acetylglucosamine
based coupling (PolyXEN, Xenetic Biosciences) [9]. (GlcNAc) stumps are then capped with galactose and
Chemically polysialylated EPO and antibody fragments NANA, resulting in simple N-glycans. [20,21]. Genome
were well tolerated in patients and had increased half- engineering of the glycosylation pathways can also result
lives [10–12]. However, glycoprotein stability, product in more defined glycosylation profiles [22].
heterogeneity and process complexity remained a prob-
lem. Apart from chemical PSA conjugation, in vitro enzy- Much more effort has gone into engineering N-glycans
matic conjugation has been demonstrated using recom- than any other type of glycan. However, a significant
binant Campylobacter jejuni Cst-II a2,8-sialyltransferase. engineering effort towards reducing heterogeneity of
This enzyme allowed for disialylation of a-1-antitrypsin mucin-type glycosylation, the most common type of O-
(A1AT) N-glycans, that could then be completely con- glycosylation in mammals, has been made as well. In
verted to PSA using Neisseria meningitidis a2,8-polysialyl- human cells, the first N-acetylgalactosamine (GalNAc)
transferase [13]. The main shortcomings of this approach residue is transferred from UDP-GalNAc to Ser or Thr
are the expensive reagents (enzymes and CMP-NANA) residues by one of 20 different GalNAc transferases.
and tightly controlled reaction conditions required to Further O-glycan synthesis usually starts with the action
prevent rehydrolysis of sialyl-units. of core 1 b1,3-galactosyltransferase (C1GalTI), which is
dependent on a specific chaperone, COSMC. A hugely
The first in vivo polysialylation strategy (Figure 2a) simplified O-glycosylation profile can be achieved by
introduced 35 NANA residues on a ScFv fusion, knocking out COSMC (‘SimpleCell’ technology).

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20 Pharmaceutical biotechnology

Figure 2

hCMAS hNANS hGNE

CST
n = ± 40

α-2,8
CMP α-2,8
α-2,6 α-2,6

hGalT hST6
N N N

hST8SiaII/IV

n = ±35

ScFv Ig5 FN1 ScFv Ig5 FN1

(a) Golgi of engineered ∆XTFT N. benthamiana and HEK293S cells

n = ±10
α-2,8
n=±3
α-2,8 α-2,8
α-2,3 α-2,3
β-1,4 β-1,4 β-1,4
ApNGT β1 LgtB β1
Cstll β1 PolyST β1
N N N N N

(b) Cytoplasm E. coli

Current Opinion in Biotechnology

Engineering strategies to allow for polysialylation of therapeutic proteins.


(a) In HEK293S cells, a polysialyltransferase and chain-initiating a2,8-sialyltransferase (PST/ST8SiaIV) were overexpressed to polysialylate the
immunoglobulin 5 (Ig5) domain of neural cell adhesion molecule (NCAM), which is fused to the protein of interest (ScFv) and to a docking domain
for the PST/ST8SiaIV (FN1). Approximately 35 NANA residues could be linked to the complex-type glycosylated (R) Ig5. In xylosyltransferase and
fucosyltransferase knockout (DXTFT) N. benthamiana cells, human UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (hGNE),
human NANA-phosphate-synthase (hNANS), human CMP-NANA synthetase (hCMAS), CMP-NANA transporter (CST), human b1,4-
galactosyltransfease (hGalT) and human a2,6-sialyltransferase (hST6) were stably overexpressed. Transient coexpression of human a2,8-
sialyltransferase (ST8SiaII/IV) in this background allowed to produce recombinant proteins containing PSA structures with a polymerization degree
of 40. (b) In E. coli, proteins could be glucosylated by the Actinobacillus pleuropneumonia cytoplasmic N-glycosyltransferase (ApNGT) and further
modified with Gal and NANA with a b1,4-galactosyltransferase from N. meningitides (LgtB) and an a2,3/a2,8-oligosialyltransferase from
Campylobacter jejuni (CstII). Recombinant proteins herein expressed had a PSA polymerization degree higher than 10.

Originally developed to sequence-map the human and number of protein-O-GalNAc transferases, CRISPR-Cas
CHO O-GalNAc glycoproteome [23], it could prove knockout is likely feasible.
useful in the future for homogenizing O-glycans on ther-
apeutics. However, the resulting single GalNAc residues In vitro glycoengineering approaches have also made it
may have immunogenic potential, as such O-glycan trun- possible to obtain highly homogeneous glycosylation, for
cations on proteins like MUC1 generates neo-epitopes. example to study IgG–FcgR interactions. For such pur-
Alternatively, as each cell type only expresses a limited poses, several companies offer a portfolio of well

Current Opinion in Biotechnology 2019, 60:17–28 www.sciencedirect.com


Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 21

characterized enzymes and nucleotide sugars (e.g.: Since FUT8 has limited activity on Man5–9 N-glycans,
Roche, CustomBiotech) [24]. Clearly, in vitro glycan these are also largely afucosyl glycans. The a-D-manno-
(re-)construction has become economically feasible sidase inhibitors kifunensine and deoxymannojirimycin
for research purposes but we have yet to see its have therefore been used to block the N-glycosylation
industrial application in biopharmaceutical manufactur- pathway at the Man8 or Man9 stage, or GnTI / to block
ing, in the light of its relatively high cost and process at the Man5 stage, all decreasing core fucosylation in
complexity [25]. CHO cells (Figure 3b-8) [34–36]. Oligomannose glyco-
proteins are expected to be rapidly cleared through the
mannose receptors (MR), which could possibly make
Antibody glycoengineering them less useful as biopharmaceuticals. However, results
Glycoengineering for altered effector functionality: in this field are controversial [35,37].
afucosylated and aglycosylated IgG
N-glycans linked to Asn297 of the Crystallizable Frag- CHO cells are currently the ‘workhorse’ for mAb produc-
ment (Fc) of IgG monoclonal antibodies (mAbs) strongly tion. Nevertheless, yeasts and plants are gradually being
influence binding to Fcg receptors (FcgR) on immune introduced as alternatives. Yeasts naturally produce afu-
cells and complement proteins, thus modulating their cosyl glycans and glycoengineered Komagataella phaffii
biological activity. Decrypting the ‘code’ of these inter- (Pichia pastoris) producing afucosylated humanized com-
actions to some extent [26], has enabled effector func- plex or oligomannose N-glycans has been developed for
tion-adapted glycoengineering. Antibody-Dependent antibody production (Merck/GlycoFi and GlycoSwitch)
Cellular Cytotoxicity (ADCC)-based therapies have been [38,39]. However, completely inactivating the endoge-
improved by the discovery that antibodies with Fc gly- nous N-glycosylation and O-glycosylation pathways with-
cans lacking a core a1,6-fucose (Fuc) (‘afucosylated anti- out affecting cell growth remains challenging, because
bodies’) exhibit an increased effector function [27]. They these glycans are a functionally important part of the yeast
bind much stronger to FcgRIIIa on Natural Killer cells cell wall. While ‘humanized’ strains produce mostly
(NK), triggering activation. Afucosylated mAbs outcom- human-type N-glycans, a certain fraction of yeast struc-
pete fucosylated human IgGs in in vitro FcgRIIIa binding tures remains. This is often overlooked as it only becomes
assays and also seem to enhance Antibody-Dependent clear upon thorough analysis. Such problems can be
Cellular Phagocytosis (ADCP) in vivo. These enhanced partially overcome through stacking of multiple glycosyl-
functionalities are important for treating tumors [28,29] transferase knockouts, but this is highly complex [40].
and possibly also to clear virally infected cells [30]. Research is ongoing to overcome these remaining chal-
lenges and it is likely that a feasible, scalable solution will
In CHO cells, afucosylated antibody production technol- soon be available (Callewaert lab, unpublished results).
ogy interferes with the GDP-Fuc and glycan biosynthesis Recently reported 2nd generation glycan ‘humanization’
pathways (Figure 3). The first afucosyl antibody (Moga- in insect cells, is also highly promising [41].
lizumab-kpkc) for treatment of relapsed or refractory
CCR4+ adult T-cell leukemia/lymphoma was approved As mentioned before, also plants have been glycoengi-
in Japan in 2012. It is produced in a CHO core-a1,6- neered (i.e. DXTFT). As an example, Lemna minor
fucosyltransferase (FUT8) knock-out cell line (Kyowa DXTFT produces antibodies with GlcNAc2Man3Glc-
Hakko Kirin, POTELLIGENT1, Figure 3b-4). Benra- NAc2 glycans, which show enhanced ADCC as compared
lizumab (also POTELLIGENT1), was approved by to fucosylated CHO produced antibody [42].
FDA in 2017. Interfering with GDP-Fuc biosynthesis
is an alternative strategy used in GlymaxX1 technology Completely removing glycans is a radically different
(ProBioGen, Figure 3b-3). In yet another approach, approach to circumvent elaborate glycoengineering strat-
Roche-GlycArt (GlycoMAb1) overexpressed N-acetyl- egies. Apart from homogeneity, simplified downstream
glucosaminyltransferase III (GnTIII) to produce glycans processing and the possibility to use microbial
carrying a bisecting GlcNAc, inhibiting core fucosylation. manufacturing make aglycosylated antibodies attractive
Obinutuzumab, an anti-CD20 mAb produced with Gly- [43]. This is particularly true when no Fc effector func-
cArt technology, shows enhanced FcgRIIIa binding, tionality is required and when the Fc mostly provides a
ADCC and antitumor activity. It also binds a different dimerization and pharmacokinetics-enhancing platform
epitope on CD20 than its comparator antibody (Ritux- for the antigen-binding domains. Indeed, importantly,
imab), making it less straightforward to assign the FcRn binding (which mediates IgG’s very long circula-
enhanced efficacy solely to glycosylation engineering tory half-life) is Fc glycosylation-independent. Initial
[31]. Further optimization encompassed co-overexpres- concerns over stability and immunogenicity have largely
sing the Golgi a-mannosidase together with GnTIII, subsided after several aglycosylated antibodies have
resulting in complex-type afucosylated bisected glycans entered the clinic without showing problems related to
(Figure 3b-7) [32]. Many other afucosylated antibodies stability or immunogenicity so far [44]. The furthest
are now in clinical trials [33]. advanced of these non-glycosylated antibodies, further

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22 Pharmaceutical biotechnology

Figure 3

(a)
CHO cell glycosylation
GDP- GMD GDP
FX
SLC35C1

GDP

GDP GDP

ManI GnTI FUT8 Manll GnTII GaIT R= RR

Golgi
Cytoplasm

(b)

Glyco-engineering strategies GDP-


3 RMD

GKDM
GDP 1 GMD GDP- 5-alkynyl-fucose
2 FX 6
5 2-fluorofucose
SLC35C1

8 DMJ
Kifunensine GDP-
7 R=
GDP GDP
ManI 9 GnTI 4 FUT8 Manll GnTII GaIT 8-9 R=

RR
GnTIII+ 1-2-3 R= ADCC
7 4-5-6
ManII FcyIIIa
Glycans resulting from different stratagies NK ceII

Golgi

Cytoplasm

Current Opinion in Biotechnology

CHO cell line glycoengineering strategies for the production of antibodies with customized glycans.
The Golgi glycosylation pathway in CHO cells and a typical biantennary N-glycan as it occurs on the Fc domain of monoclonal antibodies
produced in WT CHO cells (a). This pathway has been extensively engineered (b) to inhibit or reduce antibody fucosylation in order to enhance
FcgRIIIA binding and ADCC. The different glyco-engineering strategies are numbered from 1 to 9. Enzyme knock-out is depicted in red and (over)
expression in green. The glycan structures resulting from these strategies are shown at the right side of panel b with numbering matching to the
engineering strategies. (1) GDP-mannose 5,6-dehydratase (GMD) and (2) GDP-keto-6-deoxymannose 3,5 epimerase-4-reductase (FX) are key
enzymes in de novo GDP-fucose biosynthesis. Their inactivation blocks all cellular fucosylation [74,75]. (3) The bacterial GDP-4-keto-6-
deoxymannose reductase (RMD) can be expressed in CHO cells to convert GDP-4-keto-6-deoxymannose (GKDM), an intermediate of the GDP-
fucose synthesis pathway, to GDP-rhamnose and block GDP-fucose biosynthesis (GlymaxX, ProBioGen) [76]. Knock-out of the fucosyltransferase
FUT8 (4) is at the basis of the Potelligent technology (BioWa, Lonza) [77]. Knock-out of the GDP-fucose transporter SLC35C1 (5), which transports
GDP-fucose from the cytoplasm to the Golgi, has also been reported to eliminate Fc-fucosylation [78]. Recently, metabolic inhibition strategies
with small molecules have been developed, such as 2-fluorofucose and 5-alkynyl-Fuc (SEA technology, 6) [79]. Overexpression of GnTIII and Golgi
a-mannosidase II (7) results in bisecting GlcNAc-modified, non-fucosylated glycans (GlycArt, Roche), which show enhanced ADCC and antitumor
activity. Oligomannose Fc-linked glycans also lack the core fucose and mediate enhanced ADCC. Therefore, the a-D-mannosidase inhibitors (8)
kifunensine and deoxymannojirimycin (DMJ) have been used to block the N-glycosylation pathway at the Man8 or Man9 oligomannose stage and
decrease the fucosylation in CHO cells [34,35]. GnTI / (9) CHO cells express Man5GlcNAc2-modified IgG1 with a low degree of core fucose,
which also shows higher anti-tumour activity [36].

protein-engineered for stability and to suppress FcgR generate variants with improved FcgRIIIa binding. How-
activation, is likely ALD403/Eptinezumab (Alder Bio- ever, FcgRIIIa binding affinity could only be restored to
pharmaceuticals, Bothell, WA), which is produced in P. the level of fucosylated antibodies [45]. Despite this, the
pastoris. Furthermore, yeast surface display of mutant ADCC activity turned out to be lower than for the glyco-
libraries has generated aglycosylated Fc’s that are able to sylated antibody Herceptin. The authors speculated that
engage different FcgRs both in vitro and in vivo. Directed this is possibly due to enhanced affinity for the inhibitory
evolution of the Fc part of anti-HER2 antibodies allowed to Fcg receptor, FcgRIIb [46].

Current Opinion in Biotechnology 2019, 60:17–28 www.sciencedirect.com


Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 23

Galactosylation and sialylation of mAb Fc N-glycans the inhibitory FcgRIIB on effector macrophages seem to
Contrary to afucosyl antibodies, the role of Fc galactosy- be involved [48].
lation and sialylation remains more controversial, partially
due to overall smaller effects on antibody functionality. In Since CHO cells lack a functional ST6GAL1, they do not
vitro results suggest that high Fc N-glycan galactosylation cap their glycans with a-2,6-NANA. Co-expression of
levels may enhance some effector functions [24]. On the B4GalT1 and ST6GAL1 resulted in increased Trastuzu-
other hand, decreased antibody galactosylation is associ- mab galactosylation and sialylation [49]. Alternatively, in
ated with pathogenic autoantibodies in immune diseases vitro chemical and chemoenzymatic strategies can be
such as rheumatoid arthritis [47]. It is unclear whether this used (Figure 4). Recently, Roche developed a workflow
is a consequence of disease rather than a functionally relying on B4GalT1 and ST6Gal1 for the generation of
important alteration. Some evidence supports an anti- defined Fc glycans (Figure 4a) [11]. In an entirely differ-
inflammatory effect of increased presence of terminal ent approach, sialylation of the mAbs Rituximab and
a-2,6-NANA on fucosylated IgGs when these are admin- Trastuzumab was increased by removing the Fc glycan
istrated to treat autoimmune and inflammatory diseases. with WT EndoS2 and transglycosylating a defined sialy-
The mechanisms are still under debate but reduced lated biantennary glycan onto the remaining protein-
FcgRIIIA and C1q binding and increased expression of linked (core fucosylated) GlcNAc with mutant EndoS2

Figure 4

(a)

R=
B4GALT1 ST6GAL1

UDP UDP CMP CMP

(b)

R= EndoS2 EndoS D184M

(c)

H NH
N O
O
N
EndoS2 GalT(Y289L) click chemistry
R= N3 N H
UDP- N3 UDP

H H
O
H
O N

Current Opinion in Biotechnology

Chemoenzymatic glycoengineering for the production of antibodies with customized glycans.


(a) B4GalT1 and ST6GAL1 (Roche) can be used on heterogeneous ‘bulk’ antibody glycans for in vitro galactosylation and sialylation [24,25]. (b)
Heterogeneous Fc N-glycans are first trimmed by the wild type EndoS2. The remaining protein-linked core-fucosylated GlcNAc is then used as
acceptor for the transglycosylation of the desired N-glycans from the oxazoline substrate, catalysed by EndoS D184M [50]. (c) Similarly, enzymatic
trimming with EndoS2 is used to cleave the Fc-glycans in the chitobiose core, leaving a single GlcNAc, which is further modified by the mutant
galactosyltransferase (GalT Y289L) and UDP-GalNAz. The GalNaz moiety can then be used to couple small molecule payload (red star) with an
azide cyclooctyne click chemistry reaction. This is used for antibody labelling or preparing antibody drug-conjugates [56].

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24 Pharmaceutical biotechnology

D184M (Figure 4b) [50]. Transglycosylation to generate used to generate single GlcNAc N-glycans and subse-
sialylated glycans has also been performed with WT quently transglycosylate M6P glycan oxazolines onto
EndoS, starting with P. pastoris GlycoSwitch Super- them. Most noteworthy, (mutant) EndoA is capable of
Man5-produced Trastuzumab (Biogrammatics) [51]. transglycosylating oxazolines with M6P moieties in vary-
An outstanding issue in these remodelling approaches ing numbers and positions onto GlcNAc acceptor sites
is where to source bulk quantities of the to-be-transgly- [63,64]. However, production scalability remains chal-
cosylated N-glycan structures. Chemical synthesis is now lenging. Moreover, efficacy of the resulting drug candi-
feasible for relatively limited quantities for some of the dates has not yet been tested in vivo.
desired structures [52,53,54], and a number of natural
glycoprotein sources are available (e.g. from egg yolk) In Gaucher disease, mainly macrophages are affected. In
[55]. However, synthesis or purification from a natural these cells, recombinant glucocerebrosidase for ERT is
source in sufficient quantities for synthesizing kilograms targeted to the endocytotic macrophage receptor (MR) by
of IgG (the scale required for most real-life therapeutic virtue of various oligomannose N-glycans. In Cerezyme
applications) affordably and at cGMP-compliant quality (Genzyme), presently the most used clinical product,
is likely to remain a major challenge. Significant R&D exoglycosidases are used to trim down native CHO N-
attention is required in order to make it competitive with glycans to Man3GlcNAc2. Alternative approaches include
in cellulo glyco-optimization, which has the very important the use of mannosidase inhibitors or GnTI / CHO and
advantage of only requiring the manufacturing unit pro- HEK cells. More recently, an a-1,2-mannosidase triple
cesses commonly used in the biopharmaceutical industry. knockout HEK293S cell line allowed to produce Man8/9-
GlcNAc2-modified enzymes [65]. Plants produce pauci-
In another application of Fc glycoengineering, EndoS- mannosidic N-glycans by nature. Building on this, gluco-
type enzymes generate trimmed Fc glycans, which can cerebrosidase from transgenic carrot cells (Taliglucerase
then be specifically conjugated with chemically modified a, Elelyso) is now available as an alternative to Cerezyme
sugars to enable production of Antibody Drug Conjugates for the treatment of Gaucher’s. Furthermore, a complex
and antibody labelling via click chemistry (GlycoCon- glycan deficient A. thaliana mutant [66] and Physcomitrella
nect, Synaffix, Figure 4c) [56]. patens GnTI / mutant [67] were created to produce
Man5GlcNAc2 modified lysosomal enzymes.
Glycoengineering for (subcellular) targeting
Glycans can target therapeutic proteins to their desired site Mannose binding receptors on dendritic cells, macro-
of action. The most prominent and extensively studied phages and B cells play a key role in pathogen recogni-
targets for deliverable glycoengineered proteins are the P- tion. Lectins with other glycan specificities (sialic acid-
type lectins. These Man-6-phosphate (M6P)-binding binding Siglecs and Gal-binding lectins) are expressed on
receptors (MPR) are present in the Golgi, endosomes, specific subpopulations of immune regulatory cells.
lysosomes and on the plasma membrane of many cell types. Therefore, glycosylation of protein antigens to influence
Increasing the M6P content in enzymes for replacement immune responses has been undertaken [68]. Virus-like
therapy (ERT) in various lysosomal storage diseases particles containing hepatitis B virus small envelope
enhances cellular uptake and lysosomal delivery. Over- protein (HBsAgS VLPs), engineered to contain extra
expression of an engineered GlcNAc phosphotransferase in glycans, showed an accelerated B-cell immune response
CHO cells led to 2.1–4.9-fold increased M6P levels on acid when compared to non-glycosylated HBsAgS N146Q
a-mannosidase and acid a-glucosidase (hGAA) and better mutant and WT glycosylated VLPs. Both the hypergly-
MPR binding [57]. More recently, chemical conjugation of cosylated and WT variants carried a similar type of core-
natural and synthetic M6P-containing glycans to enzymes fucosylated bisecting glycan [69]. In another study, b1,2-
for ERT are being investigated. After the first generation of xylosyltransferase from Nicotiana tabacum was co-
chemically conjugated M6P carbohydrates [58], isosteric expressed with the extracellular part of the RSV-F surface
analogues of M6P functionalized at the anomeric position protein in CHO cells. Xylosylated RSV-F resulted in
(AMFAs), have been coupled to hGAA produced in Sf9 improved vaccination efficacy in a human artificial lymph
insect [59] and CHO cells [60]. In our laboratory, boosting node model [70]. Plant glycan modifications can be aller-
the native yeast pathways that synthesize Man-Pi-6-Man genic in mammals [71], so future will tell whether such a
glycotopes resulted in production of human lysosomal strategy of enhancing immunity will be sufficiently safe to
enzymes with >85% of phosphorylated glycans. Subse- be deployed at large. Introduction of insect-type glycans
quently, removing the phosphate-capping Man residues on the Hepatitis C virus E2 protein by production in
using a newly discovered a-mannosidase, generated M6P- Drosophila S2 cells induced a high titre of broadly neu-
modified enzymes with strongly enhanced uptake in tralizing antibodies that were able to protect humanized
patient cells [61]. mice and non-human primates against HCV challenge
[72,73]. The latter two examples indicate that the use of
In a similar approach as described for antibodies, a wide non-human glycans to increase immunogenicity repre-
range of (mutant) ENGases (reviewed in Ref. [62]) is sents an interesting strategy in vaccine design, although

Current Opinion in Biotechnology 2019, 60:17–28 www.sciencedirect.com


Custom protein glycosylation for improved biopharmaceuticals Van Landuyt et al. 25

clinical development is still needed to assess safety, as number: 1S54817N and 1S11418N] and Dr. L. Meuris is a doctor assistant
of Ghent University. This work was in part funded by an ERC consolidator
always. grant ‘GlycoTarget’ [application number: 616966], by Ghent University
projects [application number: GOA01G01412 and BOF17-GOA-018] and by
FWO Vlaanderen [application number: G041417N]. The Callewaert lab is
Conclusion partly funded by VIB. We also acknowledge C. De Wachter for proofreading
Early in vivo glycoengineering focused on the production of this manuscript.
more homogeneous glycoproteins by pushing the biosyn-
thetic pathways in mammalian cells towards the desired References and recommended reading
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Conflict of interest statement 14. Chen C, Constantinou A, Chester KA, Vyas B, Canis K,
Nothing declared. Haslam SM, Dell A, Epenetos AA, Deonarain MP:
Glycoengineering approach to half-life extension of
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L. Van Landuyt and C. Lonigro hold a strategic basic research fellowship of 15. Strasser R, Stadlmann J, Schähs M, Stiegler G, Quendler H,
the Flanders Fund for Scientific Research (FWO-Vlaanderen) [application Mach L, Glössl J, Weterings K, Pabst M, Steinkellner H:

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