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Review
Glycosylation of Recombinant AnticancerTherapeutics inDifferent
Expression SystemswithEmerging TechnologiesTariq Nadeem1, Mohsin
Ahmad Khan1, Bushra Ijaz1, Nadeem Ahmed1, Zia ur Rahman1,Muhammad
Shahzad Latif1, Qurban Ali1,2, and Muhammad Adeel Rana3
Abstract
Glycosylation, a posttranslational modification, has a majorrole
in recombinant anticancer therapeutic proteins, as most ofthe
approved recombinant therapeutics are glycoproteins. Theconstant
amino acid sequence of therapeutics determines theenzymatic
activity, while the presence of glycans influencestheir
pharmacokinetics, solubility, distribution, serum
half-life,effector function, and binding to receptors.
Glycoproteinsexpressed in different expression systems acquire
their ownoligosaccharides, which increases the protein diversity.
Theheterogeneity of glycans creates hurdles in downstream
proces-sing, ultimately leading to variable anticancer therapeutic
effi-cacy. Therefore, glycoproteins require an appropriate
expression
system to obtain structurally and functionally identical
glycans,as in humans. In many expression systems, the
N-glycosylationpathway remains conserved in the endoplasmic
reticulum,but divergence is observed when the protein enters the
Golgicomplex. Hence, in recent decades, numerous approacheshave
been adopted to engineer the Golgi's N-glycosylationpathway to
attain human-like glycans. Several researchers havetried to
engineer the N-glycosylation pathway of expressionsystems. In this
review, we examine the glycosylation patternin various expression
systems, along with emerging technologiesfor glycosylation
engineering of anticancer therapeutic drugs.Cancer Res; 78(11);
2787–98. �2018 AACR.
IntroductionCancer is the second leading cause of death in
humans, devour-
ing the lives of 8.8 million people in 2015 (1, 2). In 2025,
19.3million new cases are predicted (3). This disease is
characterizedby abnormal and uncontrolled growth of cells, which
have thepotential to invade other parts of the body through
metastasis(4, 5). Currently, most common cancer treatments
includeradiotherapy, surgery, and chemotherapy. With the
advancementof technologies, efforts are being made in clinical
treatment toidentify effective state-of-the-art therapies to
replace conventionalmethods (6, 7).
Recent advances have paved the way for the development
ofrecombinant anticancer therapeutics through engineered celllines.
As anticancerous agents, these drugs improve the deliveryof immune
cells to tumor tissues, altering the tumor microenvi-ronment,
enhancing antigen priming, and facilitating effector cellactivation
and maturation (6, 7). Production of anticancer ther-apeutic
proteins as a class of drugs is dominating the drugindustry, partly
because of the high demand and partly becauseof advancements in
recombinant DNA technology (8). The mar-
ket value of protein-based drugs is growing, with a
compoundedannual growth rate of 16% compared with the
pharmaceuticalmarket growth rate of 8% (9). Among the total
approved bio-pharmaceuticals, almost 70% are glycoproteins, which
containcarbohydrate moieties gained as a posttranslational
modificationin the process of glycosylation (10–13). This
glycosylation diver-sifies the class of biopharmaceuticals. Many
functions of antican-cer glycoproteins are associated with glycan
attachments, such assolubility, pharmacodistribution,
pharmacokinetics, properstructural folding, binding to receptors,
and serum half-life (14).
The most significant anticancer therapeutic recombinant
pro-teins are mAbs, which are glycosylated in their Fc region
(15).Alteration of the composition and structure of glycans
causesconformational changes in the Fc domain of antibodies,
affectingtheir binding affinity to Fcg receptors (16, 17). This
process leadsto a change in immune effector functions, including
complement-dependent cytotoxicity, antibody-dependent cell-mediated
cyto-toxicity (ADCC), and antibody-dependent cell-mediated
phago-cytosis (18). Deglycosylation of antibodies reduces their
bindingaffinity and hence their effector functions (19, 20).
Changes in theglycoforms of therapeutic mAbs or Fc-fusion proteins
can impactthe pharmacokinetics of proteins; for example, the
negativeimpact of hypermannosylation on pharmacokinetics can
triggertheC-type lectin clearancemechanism(15, 18, 21). Inmany
cases,the terminal sugars in the glycans can affect the
pharmacokineticsof an antibody due to glycan binding to receptors
on tissues,ultimately leading to its removal from circulation. The
majorglycan receptors that remove glycoproteins are the
mannosereceptor and the asialoglycoprotein receptor (22, 23). As
boththese receptors are abundantly expressed in the liver, it is
likelythat glycoproteins with terminal mannose or galactose
residueswill be distributed predominantly in the liver and be
catabolized
1Center of Excellence in Molecular Biology, University of the
Punjab, Lahore,Pakistan. 2Institute of Molecular Biology and
Biotechnology, University ofLahore, Lahore, Pakistan. 3Department
of Microbiology, Quaid-I-Azam Univer-sity, Islamabad, Pakistan.
Corresponding Authors: Qurban Ali, Center of Excellence in
Molecular Biology,University of the Punjab, Lahore 57300, Pakistan.
Phone: 321-962-1929; E-mail:[email protected]; and Tariq Nadeem,
[email protected]
doi: 10.1158/0008-5472.CAN-18-0032
�2018 American Association for Cancer Research.
CancerResearch
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there, as shownbyWright and colleagues in the caseof an
IgGwithterminal mannose or galactose residues (24).
Development of recombinant anticancer therapeutics hasmade
substantial progress for the treatment of various solid
andhematologic tumors over the past decade (25–27). Naked mAbsare
most commonly used to treat cancer. Rituximab was the
firstrecombinant mAb approved by FDA in 1997 and finds its
ther-apeutic applications in variety of hematologic cancers
includinglymphocytic leukemia and non-Hodgkin lymphoma. It is
ananti-CD20 humanized recombinant drug that plays a pivotal rolein
B-cell malignancies (28–30). On the other hand, trastuzumabis one
of the mAbs used for the treatment of solid tumor that iscapable of
ADCC through interactions with Fcg/Rþ immune cellsubsets. It has
transformed the treatmentofHER-2–positive breastcancer (31–33).
Tumor targeted recombinant mAbs can also beconjugated to other
forms of anticancer therapy that enhancestheir efficacy by
lessening the systemic toxicities to normal cells.There are three
types conjugated mAbs: radiolabeled that areattached to
radionuclide moieties, chemolabeled that are linkedto
antineoplastic drugs, and immunotoxin mAbs that are associ-ated
with bacterial and plant toxins (34–36). Revolution inrecombinant
DNA technology has facilitated the progress towardmore specific and
less toxic anticancer therapy (29).
In eukaryotic organisms,N-glycosylation is the most
prevalenttype of glycosylation, in which a preassembled
oligosaccharide istransferred onto asparagine (Asn) in the
consensus sequence ofthe nascent protein. This oligosaccharide
processing and matu-ration occurs regardless of the protein
template (12, 37–40).Hence, cancer glycoproteins expressed in
different expressionsystems acquire glycans depending on their own
glycosylationmachinery. Glycoproteins expressed in yeast show
hypermanno-sylated glycans, which compromise their therapeutic
efficacy(41–45). Similarly, glycoproteins expressed in plant cells
acquirexylose residues on their glycans, which show similar results
as atherapeutic agent (46, 47). Until recently, mammalian cells
hada prominent role in glycoprotein production, but alterations
intheir glycosylation pathway to produce human-like glycans
areneeded. Therefore, glycans, being an important
protein-qualityattribute, required a humanized glycosylation
machinery fortheir processing.
The rapid growth of glycoproteins and the associated
financialinterest has compelled many researchers and companies to
ana-lyze glycans. In recent years, several engineering technologies
havebeen introduced that successfully engineered the
glycosylationpathways of different expression systems (Table 1).
The commongoal of all these emerging technologies is to attach
homogeneousand human-friendly glycans on therapeutic proteins to
enhance
their effector functions. This article reviewsN-glycosylation
occur-ring in different expression systems, and we have summarized
thevarious strategies adopted in different glycosylation
engineeringtechnologies.
N-glycosylation in the EndoplasmicReticulum
EukaryoticN-glycosylation occurs in two organelles, the
endo-plasmic reticulum (ER) and the Golgi complex.
Dolichol-linkedglycan precursor formation and transfer to a nascent
protein witha little bit of processing occurs in the ER, while
complete matu-ration and processing of N-linked glycans occurs in
the Golgiapparatus. N-glycosylation occurs at asparagine residue in
theconsensus amino acid sequence (Asn-X-Ser/Thr), where X can beany
amino acid, but not proline. Theprocess is initially started
andprocessed in the ER, where oligosaccharide transferase
(OT)catalyzes the transfer of glycan onto the nascent protein
(48).Secretory proteins containing signal peptides are directed
bysignal recognition particles across the membrane into the lumenof
the ER, followed by its movement into the OT-mediatedglycosylation
machinery. Glycosylation is not dependent onprotein folding or
tertiary structure. However, some evidence hasshown that secondary
structures on both sides of the Asn con-sensus sequences may help
in this enzymatic reaction (48). Thewhole process of glycosylation
is completed in the ER and Golgibodies. Glycoproteins processed in
the ER usually show homol-ogy and remain conserved in higher
eukaryotes and yeast (49).Tetradecasaccharide (Glc3Man9GlcNAc2b1)
attached to Asn-amide groups is derived from the dolichol pathway
(50). Thesynthesis of tetradecasaccharide starts on the cytosolic
face of theER by transferring GlcNAc onto the membrane-anchored
Dol-P,yielding Dol-PP-GlcNAc in a Alg7-catalyzed reaction (51).
Infurther steps, mannose residues are attached by
mannosyltrans-ferase using the substrate GDP-Man. The first five
mannoses areattached on the cytosolic side of the ER. Glycan (32)
is thenflipped onto the luminal side by the membrane spinning
flippaseRft1p (52). Recently, biochemical studies have revealed
thatflippase (Rft1p) may not be required for this process
(53–56).In the remaining steps, four mannosyltransferases and
threeglycosyltransferases catalyze the reaction using Dol-P-Man
andDol-P-Glc as substrates, adding mannose and glucose
residues,respectively (48). After the formation of
tetradecasaccharide iscompleted, OT transfers it to the Asn residue
of the nascentprotein. Attachment of tetradecasaccharide is
followed by trim-ming of two glucose residues catalyzed by
glucosidase I andglucosidase II. Proteins then enter into the
calnexin/calreticulin
Table 1. Partial list of FDA-approved glycosylated anticancer
therapeutic drugs over the past few years
Product/INN Clinical indication Approved year References
Avelumab Merkel cell carcinoma March 2017 163Durvalumab
Urothelial carcinoma May 2017 164Inotuzumab ozogamicin B-cell
precursor acute lymphoblastic leukemia August 2017 165Atezolizumab
Urothelial carcinoma and metastatic non-small cell lung cancer May
2016 146, 166Nivolumab Classical hodgkin lymphoma May 2016
167Olaratumab Soft tissue sarcoma October 2016 168Pembrolizumab
Head and neck squamous cell cancer August 2016 169, 170Daratumumab
Multiple myeloma November 2015 171Dinutuximab Pediatrics with
neuroblastoma March 2015 172, 173Elotuzumab Mutiple myeloma
November 2015 174, 175Necitumumab Metastatic squamous non-small
cell lung cancer November 2015 176Ramucirumab Gastric cancer April
2014 177, 178
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cycle for proper folding (38, 52, 57, 58). In the last step,
gluco-sidase II removes the third glucose, allowing the protein to
enterthe Golgi complex, where species- and cell-type–specific
glyco-sylation and the remaining glycosylation process occur. In
differ-ent expression systems, the N-glycosylation pathway diverges
atthis step (59). The entire process is described in Fig. 1.
N-glycosylation in the Golgi ComplexYeast Golgi
The protein glycans formed in the ER are well conserved
indifferent eukaryotes, which are progressively changed by
differentglycosyltransferases residing in the Golgi complex (60).
Thismodification is highly diverse among different organisms
and
© 2018 American Association for Cancer Research
UDP UDP UDP UDP UDP UDP UDP
Dol-P
Dol-P
Dol-PDol-P
Dolichyl-pyrophosphate
GlcNAc
Mannose
Glucose
Alg7
Dol-P Dol-P Dol-P
Alg10 Alg8 Alg6 Alg9 Alg12
Alg9
Alg3
Alg1 Alg2? Alg2? Alg11 Alg11? Rft1p
ER Lumen
Endoplasmic reticulum
Oligosaccharyltransferase
OT
Alg13/14
Cytoplasm
Figure 1.
Synthesis of precursor oligosaccharide on themembrane of
endoplasmic reticulum and transfer on the nascent protein.
Biosynthesis of oligosaccharide is catalyzedby glycosyltransferases
encoded by different ALG loci. First synthesis starts on the
cytoplasmic face of the endoplasmic reticulum, which is then
flipped into thelumen by flippase Rft1p. As tetradecasaccharides
Glc3Man9GlcNAc2 completes, OT transfer it to the Asn residue of the
nascent protein.
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within the same organism grown in different culture
conditions(61). In the yeast Saccharomyces cerevisiae,N-linked
glycan's outerchain is further extended in the Golgi complex with
mannoseresidues. The number of mannose residues can reach up to200
with the linear backbone containing up to 50 mannoseresidues linked
via a-1,6 linkages. Further branching occursthrough a-1,2 and a-1,3
linkages, resulting in hypermannosyla-tion (62, 63). Several
monosylphosphates also attach to an outerchain, giving a negative
charge to the oligosaccharides (64).Secretory and cell wall
glycoproteins are mostly hypermannosy-lated, and the glycan may
contribute up to 95% of its molecularweight. Some intracellular
glycoproteins usually escape thesemodifications and remain intact
with short glycans of 9–13mannose residues (Fig. 2; ref. 54).
The substrate Man8GlcNAc, which comes directly from theER, is
used by a-1,6-mannosyltransferase, encoded by the OCH1gene, to add
a single mannose to initiate the outer chain (65–67).No trimming of
Man8GlcNAc occurs in yeast, unlike highereukaryotes, before outer
chain initiation (64). There is no evi-dence of oligosaccharides
smaller than Man8GlcNAc for manno-sidases in the Golgi. Therefore,
the preference of OCH1 is verynarrow (68, 69) and is mostly
Man8GlcNAc in vivo as well asin vitro (70, 71). OCH1 still
initiates outer chain formation, but iffound to be correct,
a-1,3-mannose attaches to an incompletecore oligosaccharide from
the ER in vivobut shows reduced activityon the same substrate in
vitro (69, 71).
Glycan backbone outer chain elongating enzymes,
mannosyl-transferases, are divided into two gene families. They
include theVAN1, ANP1/MNN8, and MNN9 family and the MNN10 andMNN11
family. These two families provide type II membraneencoding
proteins, which are unique of all the known
Golgiglycosyltransferases in higher eukaryotes (72). Mutation in
anyof these genes can results in truncated oligosaccharide
backboneson glycoproteins (62, 73–76). Mnn9 uses the substrate
catalyzedby OCH1. Mutant mnn9 allows the addition of only one
a-1,6-linked mannose. This is followed by a-1,2-mannose, which
isbelieved to be a stop signal. As a consequence, the
elongatingchain is terminated (77, 78), and then, it resembles the
shortglycan of some intracellular glycoproteins (79–81). Data
ofmnn9, mnn8, and mnn10 show that these enzymes function ina common
pathway, where mnn9 acts before mnn8 and mnn10(82). VAN1-mutant
data revealed that this protein is involved ininternal
a-1,2-mannose branching or backbone elongation. TheVAN1 mutant
provides mnn9-like glycosaccharides (62, 64). Infurther steps, the
core backbone is decoratedwitha-1,2-mannose,a-1,3-mannose, and
mannosylphosphate containing branches.The enzymes participating in
this branching areMnn1p, whichcatalyzes the terminal a-1,3-mannose
addition. Ktr2p, ktr1p,kre2p, and Yur1p are a-1,2-transfereases.
Mnn6p is a mannosyl-phosphotransferase (83, 84).
The last crucial step in glycosylation is chain termination.
Thecontributing factors involved in chain termination are not
wellunderstood. The distinguishing factors of hypermannosylationand
hypomannosylation are still not known. Therefore, these
twoprocesses cannot be differentiated yet (64). However, it has
beensuggested that a-1,2–linked mannose decides the fate of
outerchain elongation termination as a "stop signal." The
oligosac-charides containing terminal a-1,2-linked mannose cannot
beused as a substrate by mannosyltransferase (78). However,
the"stop signal" is not the only reason for chain termination, as
someincomplete monosaccharides do not possess terminal mannose.
The extent of glycosylationof glycoproteins expressed in yeast
alsodepends on culture medium (85), culture conditions,
availabilityof substrates, and the transportation rate through the
ER andGolgi. The use of old culture compared with fresh culture
forglycoprotein expression can affect the glycan, as observed
insecreted exoglucanase (64).
Mammalian GolgiApproximately 250 glycosyltransferases transfer
sugars in the
Golgi from donor to acceptor glycans on proteins and lipids
inmammals. Up to 20 glycosyltransferases (86) are involved in
thetransfer of sialyl sugar in mammalian Golgi. Drosophila has
justone glycosyltransferase (87–89), whereas no such
glycosyltrans-ferase has been found in yeast Golgi. This is the
reason glycopro-teins expressed in yeast lack sialyl moieties in
their glycans. Thesesugars are mainly produced in the cytoplasm and
rarely in thenucleus, that is, CMP-Sia. Then, they are transported
into theGolgi lumen by multitransmembrane transporter families(90,
91). Each glycosyltransferase is used at a specific step and,hence,
is localized in specific compartments, such as the
cis-Golgi,medial-Golgi, trans-Golgi, or trans-Golgi network (92).
Once aglycoprotein is exported into the Golgi, N-linked glycans
undergoseveral processes. In these processes, glycosidases carry
out thetrimming, while glycosyltransferases transfer sugar moieties
ontothe glycan (93). After a sugar is transported, a new
intermediatesubstrate for another glycosyltransferase is created
(94). Any sugarcontaining a free hydroxyl group can be substituted
in an inter-mediate glycanand, thus,manybranch antennae are
expected (95).
Trimming of glycans occurs in the cis-Golgi, where mannosi-dase
I catalytically removes all a-1,2-linked mannose residues. Atthis
stage, three mannose residues are removed.
N-acetylglucosa-minyltransferase I (GlcNAc-TI) adds GlcNAc to the
man-a-1,3arm of Man5GlcNAc2, which is a branched structure formed
as aresult of mannosidase I activity. Later, GlcNAc-TI converts
highmannose-type glycans into a hybrid and complex type by
theaddition of GlcNAc. In the medial-Golgi, a-1,3- and
a-1,6-linkedmannose is removed by mannosidase II. GlcNAc is then
addedonto a-1,6 mannose by GlcNAc-TII. In this way, a
hybrid-typeglycan is converted into a complex-type glycan. Many
branchesthat become biantenary, triantenary, tetra-antenary, and
penta-antenary oligosaccharides can be generated by
GlcNAc-TIV,GlcNAc-TV, and/or GlcNAc-TVI. GlcNAc-TIII can prevent
theactivity of GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, andmannosidaseII
as it brings bisectingGlcNAc residue ontob-mannose of the core(93,
96). Fucosyltransferase can then add fucose residue to thevery
first GlcNAc directly attached to Asn in the polypeptide.Fucose
addition occurs in the medial-Golgi. In most of the cases,following
the fucose addition, glycoprotein is shifted to thetrans-Golgi for
terminal glycosylation. Galactose and sialic acidare attached to
eachN-glycan antenna. Galactose is usually addedby b-1,4 and b-1,3
galactosyltransferase. In the end, terminal sialicacid is added to
galactose by sialyltransferase. The most commonsialic acid in
humans is NeuAc, which is added in an a-2,3, a-2,6,or a-2,8 linkage
to galactose (93).
N-glycosylation Engineering Technologiesfor Yeast
S. cerevisiae is a robust expression system for
heterologousrecombinant drugs production. Because of possibly
higher titers,low risk of human viral contamination and low
scalable
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fermentation process, yeast-based protein production platformis
regarded as an alternative to mammalian expression system(97–99).
Mixture of two anticancer therapeutic proteins, anti-CTLA4 and
anti-PD1, has been produced in yeast. These recom-binant antibodies
act as checkpoint inhibitors approved formanagement of advanced
melanoma (100–102). PD1 and
CTLA4 regulate T cells through negative feedback mechanism,but
they are upregulated at different stages of T-cell activation.The
human anticytotoxic T-lymphocyte–associated antigen 4antibody
attaches to CTLA4 on the T-cell surface, preventingCTLA4 from
inhibiting T-cell activation, whereas the humananti-programmed cell
death 1 antibody binds to PD1, blocking
© 2018 American Association for Cancer Research
GnT I
GnT I
Gm II
GnT II
XyITFuT 11/12
GalT IFuT 13ST
Mns II
Mns 1/2
1, 2 MnTs1, 2 MnTs
*
*
ER (Human, Yeast, Plants)
Golgi (Human) Golgi (Yeast) Golgi (Plants)
Man8BGIcNAc2
Man5GIcNAc2 Man9GIcNAc2
β-1, 4-Galα-1, 2-Manα-1, 6-Manα-1, 3-Manβ-1, 4-Manβ-1,
N-GlcNAcβ-1, 4-GlcNAcβ-1, 2-GlcNAcα-2, 3-NANA/α-2, 6-NANAPresent in
S. cerevisiae butnot in P. pastroris
Xylose
Fucose
GIcNAcMan5GIcNAc2
GaI2GlcNAc2Man3GIcNAc2
NANA2GaI2GlcNAc2Man3GIcNAc2
GalT
GIcNAc2Man3GIcNAc2
GnT II
GIcNAcMan3GIcNAc2
Figure 2.
Glycosylation pathway in humans, yeast, and plants. The
representative pathway model of human is used as a template for
glycoengineering mammalian,yeast, and plant cells to obtain
humanized glycoproteins. ER, endoplasmic reticulum; GalT,
galactosyltransferase; GlcNAc, N-acetylglucosamine; GnT
I,N-acetylglucosaminyl transferase I; GnT II, N-acetylglucosaminyl
transferase II; Man, mannose; Mns II, mannosidase II; MnTs,
mannosyltransferase; NANA,N-acetylneuraminic acid; ST,
sialyltransferase; GmII, Golgi a-mannosidase II; XylT,
b1,2-xylosyltransferase; FuT11/12, core a1,3-fucosyltransferases;
FuT13,a1,4-fucosyltransferase.
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tumor cells from shutting down T-cell activity (103, 104).
Mixtureof these recombinant antibodies was produced by
coculturingtwo Pichia pastoris strains such that each produced one
ofthe mAbs under optimized culture conditions. Confirmationof
correct structures and targets of the antibodies produced inP.
pastoris was affirmed through binding and competitive assays.This
reflects production of multiple recombinant anticancertherapeutics
in P. pastoris by integrating inducible pro-tein expression
systems. Thus, we envision that this expressionsystem has the
potential to reduce time, cost, number of strains,and facilities
required for anticancer therapeutics production(105). In this
regard, various technologies have been introducedto humanized yeast
expression system (Table 2).
GlycoSwitch technologyJacobs and colleagues have successfully
engineered the
N-glycosylation pathway of the yeast P. pastoris for the
productionof humanized glycoproteins (10). In their strategy, they
knockedout a gene involved in hypermannosylation and
introducedvarious mammalian pathway genes to attain
human-likeN-glycan on recombinant glycoproteins. A total of five
Glyco-Switch vectors were introduced, step by step, containing
differentselection markers. The protocol followed is not suitable
forresolving the issue of nonhuman O-linked glycosylation inP.
pastoris (8). As described previously, the pattern of glycanremains
conserved at the ER level and diverges when it entersinto the Golgi
complex (59). To stop this hypermannosylation,the
a-1,6-mannosyltransferase OCH1 gene was disrupted, asit initiates
the outer chain leading to hypermannosylatedbranches. For this
inactivation, the pGlycoSwitchM8 vector wasused, which replaces the
actual OCH1 with a nonactive fragmentby homologous recombination.
The strain M8 produced as aresult of this inactivationwas able to
control hyperglycosylation atthe Man8GlcNAc glycan level. After
this, HDEL-tagged a-1,2-mannosidase, from Trichoderma reesei
fungus, was introduced.The resulting strain M5 successfully
modified the glycan to Man5-GlcNAc, as the introduced gene had
mostly removed all terminala-1,2–linked mannose residues (42).
To convert N-glycan into a hybrid-type, GlcNAc transferase
I(GnT-I) was introduced. For this purpose, the human GnT-Icatalytic
domain was fused with the Kre2p N-terminus domainof S. cerevisiae
(42). The Kre2p contributes to proper cis/medial-Golgi localization
(106). The resulting strain, GnM5, was able tomodify the glycan
into GlcNAcMan5GlcNAc2. The next step inengineering was to add
galactose to b-1,2-GlcNAc. For this, theGnM5 strain was transformed
with the pGlycoSwitchGalT vector.The vector had a tripartite fusion
protein. The first part, UDP-Gal4-epimerase, converts UDP-Glc into
UDP-Gal, thus ensuring itsavailability in the Golgi complex. The
second part, the catalytic
domain of human b-1,4-galactosyltransferase I,
catalyzesgalac-tose addition. For proper Golgi localization, the S.
cerevisiaedomain was included in the fusion protein Mnn2p (8)
[thisstrategy was used for the first time for Escherichia coli
(107)and GlycoFi adapted it for P. pastoris (108)]. The
resultingmodified strain GalGnM5 was able to produce a
hybrid-typeGalGlcNAcMan5GlcNAc glycan.
Furthermore, engineering of complex and hybrid-type glycanswas
carried out by introducing mannosidase II and GlcNActrans-ferase II
(GnT-II). First, the catalytic domain of mannosidase IIfrom
Drosophila melanogaster was fused with the S. cerevisiaeMnn2P Golgi
localization domain. Introduction of this fusionprotein resulted in
a GnM3 strain, which was able to removeterminal a-1,3 and
a-1,6-linked mannose. Hence, the GnM3strain modified its
glycoproteins with a GlcNAcMan3GlcNAc2-type glycan. Transforming
the fusion protein Mnn2DmMan-IIinto GalGnM5 strain resulted in a
GalGnM3 strain, which couldmodify N-glycan with a
GalGlcNAcMan3GlcNAc2 glycan struc-ture. In the very last step for
the addition of terminal galactoseonto the biantennary complex type
glycan, GnT-II was intro-duced. The catalytic domain of Rat GnT-II
was fused with theS. cerevisiae Mnn2p N-terminal domain (109). The
resultingstrain, Gal2Gn2M3, was capable of synthesizing
Gal2GlcNAc2-Man3GlcNAc-type N-glycans. Three different types of
proteins,mouse IL10, mouse GM-CSF, and mouse IL22, which
haveN-glycosylation sites that were expressed in each of
thesestrains, were produced as a result of engineering.
N-linkedhyperglycosylation was successfully controlled using
thesestrains. It was also observed that the strain that was
extensivelyengineered had increased glycan heterogeneity. This
heteroge-neity is believed to be caused by incomplete processing
andhindrance created by endogenous monosyltransferases. Thiscan be
overcome by optimizing growth conditions and aculturing medium (8).
Pichia GlycoSwitch has joined handsin December 2014 with UTV
Technologies, where they can useVTU Technology yield-enhancing P.
pastoris expression plat-form. UTV has the broadest toolbox and
versatile technologiesfor expressing recombinant proteins in P.
pastoris and hasalready achieved the target of 22 g/L of secretory
proteins. Thepartnership of both technologies can ensure the better
yield ofhumanized anticancer glycoproteins (110).
GlycoFi technologyIn an attempt to humanize the glycosylation
pathway of yeast
for humanized glycoproteins, GlycoFi Inc. was founded by
Pro-fessor Gerngross and Professor Hutchinson in 2000. In
GlycoFitechnology, a total of four genes of P. pastoris were
knocked out,and14geneswere introduced. Consequently, themodified
strainscould produce more than 90% homogenous glycoproteins
with
Table 2. Various glycoengineering technologies
Company Glyco Technology Cell type Drugs/protein
VTU/RCT GlycoSwitch Pichia pastoris (Yeast) GM-CSF, CH2, IL22
Domain, IL10, IFNb, TransferrinGlycode (FR) GlycodExpress
Saccharomyces cerevisiae (Yeast)Merck (US) GlycoFi Pichia pastoris
(Yeast) EPOGlycotope GlycoExpress Human cell lines EGFR, HER2
GlycoDelete HEK GM-CSF, anti-CD20siRNA mediated glycoengineering
CHO (hamster) IgG1
Kyowa Hakko Kirin (JP) Lonza (UK) POTELLIGENT CHO (hamster)
CCR4, CD98, GM2, IL5Roche-Glycart (CH) GlycoMAb CHO (hamster) CD20,
EGFR, HER2, HER3
Abbreviations: CCR4, C-C Chemokine receptor type 4; CD, Cluster
of differentiation; CH, Switzerland; CHO; Chinese hamster ovary;
EPO, epidermal growthfactor; FR, France.
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complex N-glycans, similar to humans. The genes introducedmostly
consisted of catalytic domains from mammalian originand signal
peptides from fungi for proper Golgi localization(111). To modify
the P. pastoris N-glycosylation pathway, theresearchers performed
the following steps. At first, they knockedout the OCH1 (endogenous
mannosyltransferase gene) using thegene disruption method (111).
For efficient disruption, variousvectors were designed; these
vectors contained resistance genesfor selection or selection was
made on the basis of auxotrophy(112, 113). TheOCH1 gene is involved
inhyperglycosylation, as itprovides the outer branch for further
glycosylation. After genedisruption, UDP-Gal and UDP-GlcNAc
transporters were intro-duced, which ensured the availability of
sugar precursors in theGolgi complex. At last, genes of mammalian
origin involved intrimming and addition of sugar moieties such as
glucanases andglycosyltransferases were introduced (111, 114,
115).
Erythropoeitin (EPO) is a very important therapeutic
glyco-protein consisting of 165 amino acids. This protein has three
N-glycosylation (Asn24, 38, 83) sites (116), which have amajor role
inits activity, secretion, and bioactivity in various types of
cancer(117). Removal of N-glycan by mutagenesis resulted in a
sub-stantial decrease in bioactivity, stability, and hindrance in
secre-tion. Similarly, N-glycans with no terminal sialic acids have
theirgalactose exposed and are easily removed by
galactose-specificreceptors in serum (118). EPO, produced via
GlycoFi technology,has a human-like glycan and, when compared with
EPO, has ayeast-like glycan (highly monosylated) in rat, showing
remark-able improvement inbioactivity and serumhalf-life
(119).Hence,the glycoproteins produced viaGlycoFi technologywere
shown tohave an advantage over those produced in wild-type yeast or
lessengineered yeast.
N-glycosylation Engineering Technologiesfor Mammalian Cells
Human cell lines allow human-like glycosylation of recombi-nant
anticancer proteins. This approach warrants that proteinsharbor at
least nonimmunogenic glycans even then a lot ofpromising
technologies are being introduced to humanizedrecombinant
therapeutics glycosylation (120). FDA has approvedmany recombinant
anticancer therapeutics produced in ChineseHamster Ovary (CHO)
cells (121, 122). Among these, pertuzu-mab (HER2 dimerization
inhibitor), daratumumab (CD38-tar-getedmAb), rituximab
(anti-CD20mAb), and siltuximab are justsome of the many examples
used to treat breast cancer, relapsedmultiple myeloma, non-Hodgkin
B-cell lymphoma, and idio-pathic multicentric Castleman disease,
respectively (122–124).Among human cell lines, the HT-1080 (having
fibrosarcomaorigin) and the HEK293 (derived from human embryo
kidney)cells are used to manufacture glycosylated recombinant
thera-peutics. Agalsidase alfa, velaglucerase alfa rFVIIIFc,
rFIXFc, epoetindelta, and idursulfase are some of the many
therapeutics pro-duced in human cell lines. Additional recombinant
therapeuticsproduced in theHuH-7 (hepatocellular carcinoma cells),
HKB-11(Kidney/B Cell Hybrid), PER.C6 (Crucell), and CAP
(CEVECAmniocyte Production) human cell lines are currently
beingexamined (125–127). Murine myeloma cell lines (NS0 andSp2/0)
have also been used for the production of recombinantanticancer
mAbs such as elotuzumab (SLAMF7-directed immu-nostimulatory
antibody), cetuximab (inhibits EGFR), dinutuxi-mab (chimeric
antibody), ofatumumab (anti-CD20 mAb), and
necitumumab (EGFR antagonist) that are active in treatmentof
multiple myeloma, colorectal cancer, neuroblastoma,
chroniclymphocytic leukemia, and nonsmall cell lung cancer
(128–132).Some of the emerging mammalian cell line technologies
arediscussed below.
Mammalian cells' engineering via siRNAHuman IgG1 isotype
contains two Asn-linked glycosylation
sites in its Fc region (133). Fc- mediated effector function
isinfluenced by the N-glycan attached to it (134, 135). As
studieshave shown, core fucose lacking glycan of the Fc region
ofantibodies exhibits more efficiency than fucosylated
antibodies,both in vivo and in vitro (136–140). Unfortunately,
almost allavailable therapeutic antibodies on the market are highly
fuco-sylated, mostly containing fucose in their core
oligosaccharide. Inmammalian cell lines, the fucosylation of
glycoproteins is medi-ated by the a-1,6-fucosyltransferase (FUT8)
gene, which transfersfucose residue from GDP-fucose to GlcNAc in
the N-glycan ofglycoprotein (141). The substrate (GDP-fucose) of
glycan fuco-sylation is manufactured in the cytoplasm by both de
novo andsalvage pathways. The de novo pathway, which contributes
tomostof the intracellular GDP-fucose, has an enzyme,
GDP-mannose4,6-dehydrate (GMD), involved in enzymatic reaction of
thepathway. The enzymes FUT8 and GMD can be important candi-dates
in controlling fucosylation of oligosaccharides (142, 143).
In a study carried out by Imai-Nishiya and colleagues
(111),antibodies producing CHO cell lines to nonfucosylated
antibo-dies producing cells have been engineered without disturbing
anycharacteristics of cells, except fucosylation. In their
strategy, theresearchers used RNA interference with Lens culinaris
agglutinin(LCA) lectin as a phenotypic selection strategy. LCA
lectin recog-nizes the a-1,6-fucosylated trimannose glycan core in
cells andcommits them to apoptosis. For knockdown of the genes
GMDand FUT8, constitutive vectors expressing siRNA against
thesegenes were introduced into an antibody producing
CHO/DG4432-05-12 cells (144, 145). Clones expressing a low level
oftargeted genes were selected for antibody analysis, which
showedalmost no fucosylation. They concluded that this strategy
forcontrolling fucosylation of antibodies to enhance ADCC is
quiteeffective, economical, and less time consuming compared
withthe use of homologous recombination for gene targeting
inmammalian somatic cells (145). This strategy has the potentialfor
the development of next-generation antibodies for
anticancertherapeutic use (146).
GlycoExpress technologyGlycotope GmbH, founded in 2001,
developed novel technol-
ogies for production of biopharmaceuticals and then focused onan
expression system that produces fully humanized glycopro-teins. For
this purpose, they developed GlycoExpress technology(95), based on
mammalian cell lines, which can produce andoptimize humanized
glycoproteins (147). Most of the mamma-lian cell lines (e.g., CHO,
BHK, or SP2/O) used for anticancertherapeutics production can
produce glycoproteins with glycanssimilar to those of humans but
lacking a few important moieties,just as a-2,6-linked
sialylationand bisecting GlcNAc are missingin these glycoproteins.
On the contrary, few nonhuman addition-al moieties are present,
such as terminal NeuGc (a type of sialicacid) or galactose attached
to another galactose at the terminalposition (148). These extra
nonhuman components can increasethe immunogenic response (149). To
solve these problems,
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Glycotope used human cell lines in its GlycoExpress
technologyand further engineered them, as proteins are not
glycosylated inthe same way in different types of cells. Different
sets of cell lineswere formed to achieve different glycan
profiles.
GlycoExpress cell lines are engineered using various
techni-ques. The gene that needs to be removed is knocked out via
gene-specific recombination, and then, the stable cell lines are
isolatedafter transfection with various glycosylation enzymes.
Cells engi-neered in the GlycoExpress toolbox can then address
differentsteps of glycosylation. They can produce fucosylated or
nonfu-cosylated, a-2,3 and a-2,6-sialylated or nonsialylated,
highlygalactosylated, or nongalactosylated, and ranging from
hybridto complex-type glycoproteins. Glycoproteins with greater
serumhalf-lives can be produced in these cells by sialylating them
to veryhigh degrees. Similarly, cell lines are available that can
fucosylateor sialylate glycoproteins in the range of 0% to
naturally possiblemaximum.However, this canbe achievedbyoptimizing
culturingconditions andmedium supplements (150–152). Antibodies
arethe most important class of biotherapeutics and the major
targetfor glycol optimization. When IgG1 antibodies were produced
inGlycoExpress cell lines, a 10- to250-fold increase was found in
itsADCC activity to improve anticancer therapy compared withthose
originally produced in rodent cell lines. Similar results werefound
with other types of antibodies produced in GlycoExpresscell lines.
In most cases, these results were obtained by fucoseremoval,
addition of bisecting GlcNAc, and a high degree ofgalactosylation
and sialylation. In one study, one type of antibodyhad enhanced
ADCC activity without fucose removal (147). Thisstudy demonstrates
that the perception that a-1,6-fucosylationremoval is the onlyway
to improve ADCC to treat various types ofcancers is incorrect.
GlycoDelete technologyFor efficient activity of glycoproteins,
theyneedhumanized and
homogenous glycans (153). The heterogeneity of glycans invarious
expression systems is due to different steps of complex-type
N-glycan production. To achieve homogenous glycan pro-duction,
Meuris and colleagues introduced a technology calledGlycoDelete.
This technology simplified and shortened themam-malian
N-glycosylation pathway, leading to proteins with shortand simple
glycan carrying a sialylated trisaccharide. Cells pro-duced as a
result of GlycoDelete technology did not lose normalphysiologic
processes or protein folding due to glycan modifica-tion (154).
Meuris and colleagues started GlycoDelete engineer-ing from human
embryonic kidney 293S. These cells were defi-cient in carrying out
N-acetylglucosaminyl transferase I (GnTI)activity, which converts
N-glycan into hybrid and complex typeglycan. GnTI-mutant cells
[293SGnTI (�) cells] were previouslyproduced by deleting GnTI
(155). Then, these cells were trans-fected with the fusion
protein–containing fungus Hypocrea jecor-ina endoT
(endo-b-N-acetylglucosaminidases; ref. 122) catalyticdomain and the
human b-galactoside-a-2,6-sialyltransferase I(ST6GALI; ref. 123)
targeting domain for proper Golgi local-ization. Endo T removes
N-linked oligosaccharides and leavesthe glycoprotein with a single
GlcNAc by breaking bondsbetween the first two GlcNAc residues
(156). Then, this struc-ture is recognized by
galactosyltransferases and sialyltrans-ferases, adding up galactose
and sialic acid, respectively.Because of this GlycoDelete strategy,
the N-glycosylation path-way remains confined to a three-step
process, which success-fully homogenizes N-glycan (154).
For stable modified cell line isolation, Concanavalin A (157)was
used for selection, which recognizes mannosylated, hybridand
complex type N-glycans on cell surface proteins, leavingbehind
GlycoDelete phenotypes. Later, it was found that 293SGlycoDelete
cells are less adherent, which is favorable for sus-pension
culturing (154). GM-CSF (158) and anti-CD20 wereexpressed in 293S
GlycoDelete lines for N-glycan analysis. Thesecells were found to
produce sialated trisaccarides or Gal-GlcNAcdisaccharides and
rarely monosaccharide intermediates in con-trast to complex- and
hybrid-type N-glycan by other types ofmammalian cell lines. Meuris
and colleagues also discovered thatGlycoDelete anti-CD20 antibodies
have a greater initial serumhalf-life than wild-type anti-CD20 in
mice. This finding may bedue to a decrease in sialated
glycoproteins binding to lectinreceptors, which can lead to its
clearance from the serum(159). Similarly, GlycoDelete antibodies
showed more than10-fold decrease in binding affinity to FcRs of
humans, which isgood, as safety is a concern in the case of
neutralizing antibodies(159). In the case of antibodies binding to
these receptors, theimmune response is evoked and cytokine
production is triggered.Therefore, GlycoDelete technology favors
production of anti-bodies when the case is neutralization of
antigen rather thanadditional effector function. Likewise, the
reduced N-glycanpro-tocol is important in the biopharmaceutical
industry, and thebenefits of short N-glycans have also been
reported (160–162).
Conclusion and Future ProspectsGlycosylation is the most
frequent posttranslational modifica-
tion of anticancer therapeutic proteins and therefore has a
majorinfluence on biologic activity, specificity, and complexity,
makingthem less immunogenic and well-tolerated. The safety profile
andhigh efficacy of these drugs has resulted in incredible growth
inalmost every area ofmedicine. Advancement of unique
expressiontechnologies, such as process optimization, modified
hosts, pro-moters, and secretion signals, has facilitated
production of gramquantities of anticancer recombinant drugs at low
cost andwithina short periodof time. Among a variety of expression
systems (e.g.,yeast and mammalian cell lines) currently employed
for produc-tion of glycosylated anticancer therapeutics,
mammalian-basedsystems have been predominantly used. The major
contributingfactors in selection of the expression system are the
glycosylationcomposition and glycoforms or patterns. Anticancer
glycopro-teins produced via GlycoFi technology in yeast showmore
resem-blance to their natural counterparts than those produced in
lessengineered or wild-type yeast. Glycosylated anticancer
therapeu-tics such as antibodies produced through GlycoExpress
technol-ogy possess many folds increase in ADCC activity compared
withany other glycoengineering technology. Furthermore,
antibodiesproduced through GlycoDelete technology have a greater
serumhalf-life and decreased binding affinity to humanFcRs,
whichenhances their safety profile.
Despite the increasing number of glycosylation
engineeringtechnologies, along with their expression systems
available foruse, there is no technology capable of meeting all
challenges.Different glycosylation parameters (e.g., the glycan
charge,sequence, molecular size, and number of glycans attached)can
modulate the emerging technologies used in differentexpression
systems to different extents in the near future. Thesignificant
potential of these technologies in different expres-sion systems
should lead to further research toward the
Nadeem et al.
Cancer Res; 78(11) June 1, 2018 Cancer Research2794
on June 14, 2020. © 2018 American Association for Cancer
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development of anticancer therapeutic drugs with the
lowestprobability of contamination, high yield, inexpensive
medium,human-like glycan isoforms, improving delivery of
immunecells to tumor tissues, increasing antigen priming, and
facili-tating effector cell activation.
Disclosure of Potential Conflicts of InterestNo potential
conflicts of interest were disclosed.
Received January 18, 2018; revised March 22, 2018; accepted
April 3, 2018;published first May 22, 2018.
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