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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.

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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.

Anticancer Therapeutics in Different Expression Systems

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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.






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,






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.





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.

References1. Reddy KS. Global Burden of Disease Study 2015 provides GPS for global

health 2030. Lancet 2016;388:1448–9.2. Mokdad AH. Burden of lower respiratory infections in the Eastern

Mediterranean Region between 1990 and 2015: findings fromthe Global Burden of Disease 2015 study. Int J Public Health 2017;60:1–12.

3. International Agency for Research on Cancer. GLOBOCAN 2012: Esti-mated cancer incidence, mortality, and prevalence worldwide in 2012.Lyon, France: IARC; 2012. Available from: http://globocan.iarc.fr/Default.aspx.

4. Hirschey MD, DeBerardinis RJ, Diehl AME, Drew JE, Frezza C, Green MF,et al. Dysregulatedmetabolism contributes to oncogenesis. Semin CancerBiol 2015;35:S129–S50.

5. Bhattacharya S, Ghosh MK. Cell death and deubiquitinases: perspectivesin cancer. BioMed Res Int 2014;2014:435197.

6. LechnerMG, Russell SM, Bass RS, Epstein AL. Chemokines, costimulatorymolecules and fusion proteins for the immunotherapy of solid tumors.Immunotherapy 2011;3:1317–40.

7. Conibear AC, Schmid A, Kamalov M, Becker CFW, Bello C. Recentadvances in peptide-based approaches for cancer treatment. Curr MedChem 2017. doi: 10.2174/0929867325666171123204851.

8. Jacobs PP, Geysens S, Vervecken W, Contreras R, Callewaert N. Engineer-ing complex-type N-glycosylation in Pichia pastoris using GlycoSwitchtechnology. Nat Protoc 2009;4:58–70.

9. EvaluatePharma. World Preview 2016, Outlook to 2022; 2016. Availablefrom: http://info evaluategroup com/rs/607-YGS-364/images/wp16 pdf.

10. Wildt S, Gerngross TU. The humanization of N-glycosylation pathways inyeast. Nat Rev Microbiol 2005;3:119–28.

11. Sethuraman N, Stadheim TA. Challenges in therapeutic glycoproteinproduction. Curr Opin Biotechnol 2006;17:341–6.

12. Veillon L, Fakih C, Abou-El-Hassan H, Kobeissy F, Mechref Y. Glycosy-lationchanges in brain cancer. ACS Chem Neurosci 2018;9:51–72.

13. Mizukami A, Caron AL, Picanco-Castro V, Swiech K. Platforms forrecombinant therapeutic glycoprotein production. Methods Mol Biol2018;1674:1–14.

14. Wang LX, Lomino JV. Emerging technologies for making glycan-definedglycoproteins. ACS Chem Biol 2012;7:110–22.

15. Liu L. Antibody glycosylation and its impact on the pharmacokineticsand pharmacodynamics of monoclonal antibodies and Fc-fusion pro-teins. J Pharm Sci 2015;104:1866–84.

16. Krapp S,Mimura Y, Jefferis R, Huber R, Sondermann P. Structural analysisof human IgG-Fc glycoforms reveals a correlation between glycosylationand structural integrity. J Mol Biol 2003;325:979–89.

17. Arnold JN, Wormald MR, Sim RB, Rudd PM, Dwek RA. The impact ofglycosylation on the biological function and structure of human immu-noglobulins. Annu Rev Immunol 2007;25:21–50.

18. Liu L, Gomathinayagam S, Hamuro L, Prueksaritanont T, Wang W,Stadheim TA, et al. The impact of glycosylation on the pharmacokineticsof a TNFR2: Fc fusion protein expressed in glycoengineered Pichia pastoris.Pharm Res 2013;30:803–12.

19. Tao MH, Morrison SL. Studies of aglycosylated chimeric mouse-humanIgG. Role of carbohydrate in the structure and effector functionsmediatedby the human IgG constant region. J Immunol 1989;143:2595–601.

20. Sazinsky SL, Ott RG, Silver NW, Tidor B, Ravetch JV, Wittrup KD.Aglycosylated immunoglobulin G1 variants productively engage activat-ing Fc receptors. Proc Natl Acad Sci U S A 2008;105:20167–72.

21. Goetze AM, Liu YD, Zhang Z, Shah B, Lee E, Bondarenko PV, et al. High-mannose glycans on the Fc region of therapeutic IgG antibodies increaseserum clearance in humans. Glycobiology 2011;21:949–59.

22. Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. AnnuRev Biochem 1982;51:531–54.

23. Mi Y, Lin A, Fiete D, Steirer L, Baenziger JU. Modulation of mannose andasialoglycoprotein receptor expression determines glycoprotein hormonehalf-life at critical points in the reproductive cycle. J Biol Chem 2014;289:12157–67.

24. Wright A, SatoY,Okada T,ChangK, EndoT,Morrison S. In vivo traffickingand catabolism of IgG1 antibodies with Fc associated carbohydrates ofdiffering structure. Glycobiology 2000;10:1347–55.

25. Redman JM, Hill EM, AlDeghaither D, Weiner LM. Mechanisms of actionof therapeutic antibodies for cancer. Mol Immunol 2015;67:28–45.

26. Honeychurch J, Cheadle EJ, Dovedi SJ, Illidge TM. Immuno-regulatoryantibodies for the treatment of cancer. Expert Opin Biol Ther 2015;15:787–801.

27. Coulson A, Levy A, Gossell-WilliamsM.Monoclonal antibodies in cancertherapy: mechanisms, successes and limitations. West Indian Med J2014;63:650–4.

28. Oldham RK, Dillman RO. Monoclonal antibodies in cancer therapy: 25years of progress. J Clin Oncol 2008;26:1774–7.

29. Zhou X, Lisenko K, Lehners N, Egerer G, Ho AD, Witzens-Harig M.The influence of rituximab-containing chemotherapy on HCV load inpatients with HCV-associated non-Hodgkin's lymphomas. Ann Hematol2017;96:1501–7.

30. Chaoui D, Choquet S, Sanhes L, Mahe B, Hacini M, Fitoussi O, et al.Relapsed chronic lymphocytic leukemia retreatedwith rituximab: interimresults of the PERLE study. Leuk Lymphoma 2017;58:1366–75.

31. Kordbacheh T, Law WY, Smith IE. Sanctuary site leptomeningeal metas-tases in HER-2 positive breast cancer: a review in the era of trastuzumab.Breast 2016;26:54–8.

32. Richard S, Selle F, Lotz JP, Khalil A, Gligorov J, Soares DG. Pertuzumaband trastuzumab: the rationale way to synergy. An Acad Bras Cienc2016;88:565–77.

33. Collins DM, Gately K, Hughes C, Edwards C, Davies A, Madden SF, et al.Tyrosine kinase inhibitors as modulators of trastuzumab-mediated anti-body-dependent cell-mediated cytotoxicity in breast cancer cell lines. CellImmunol 2017;319:35–42.

34. Kawashima H. Radioimmunotherapy: a specific treatment protocol forcancer by cytotoxic radioisotopes conjugated to antibodies. ScientificWorld J 2014;2014:492061.

35. Zhang N, Zhang J, Wang P, Liu X, Huo P, Xu Y, et al. Investigation of anantitumor drug-delivery system based on anti-HER2 antibody-conjugat-ed BSA nanoparticles. Anticancer Drugs 2018;29:307–22.

36. Scott AM, Allison JP, Wolchok JD. Monoclonal antibodies in cancertherapy. Cancer Immun 2012;12:14.

37. Aebi M, Bernasconi R, Clerc S, Molinari M. N-glycan structures: recogni-tion and processing in the ER. Trends Biochem Sci 2010;35:74–82.

38. Helenius A, Aebi M. Roles of N-linked glycans in the endoplasmicreticulum. Annu Rev Biochem 2004;73:1019–49.

39. Larkin A, Imperiali B. The expanding horizons of asparagine-linkedglycosylation. Biochemistry 2011;50:4411–26.

40. Schwarz F, Aebi M. Mechanisms and principles of N-linked proteinglycosylation. Curr Opin Struct Biol 2011;21:576–82.

41. Krainer FW,Gmeiner C,Neutsch L,WindwarderM, Pletzenauer R,HerwigC, et al. Knockout of an endogenous mannosyltransferase increases thehomogeneity of glycoproteins produced in Pichia pastoris. Sci Rep2013;3:3279.

42. Vervecken W, Kaigorodov V, Callewaert N, Geysens S, De Vusser K,Contreras R. Invivo synthesis of mammalian-like, hybrid-type N-glycansin Pichiapastoris. Appl Environ Microbiol 2004;70:2639–46.

43. Bretthauer RK. Genetic engineering of Pichia pastoris to humanizeN-glycosylation of proteins. Trends Biotechnol 2003;21:459–62.

44. Chung CY, Majewska NI, Wang Q, Paul JT, Betenbaugh MJ. SnapShot: N-glycosylation processing pathways across kingdoms. Cell 2017;171:258.





http://globocan.iarc.fr/Default.aspxhttp://globocan.iarc.fr/Default.aspxhttp://info evaluategroup com/rs/607-YGS-364/images/wp16 pdfhttp://cancerres.aacrjournals.org/

45. Wang W, Soriano B, Chen Q. Glycan profiling of proteins usinglectin binding by surface plasmon resonance. Anal Biochem 2017;538:53–63.

46. Fitchette-Laine A-C, Gomord V, Chekkaf A, Fay L. Distribution of xylo-sylation and fucosylation in the plant Golgi apparatus. Plant J1994;5:673–82.

47. Rayon C, Lerouge P, Faye L. The proteinN-glycosylation in plants. J ExpBot 1998;49:1463–72.

48. Weerapana E, Imperiali B. Asparagine-linked protein glycosylation: fromeukaryotic to prokaryotic systems. Glycobiology 2006;16:91R–101R.

49. Gemmill TR, Trimble RB. Overview of N- and O-linked oligosaccharidestructures found in various yeast species. Biochim Biophys Acta 1999;1426:227–37.

50. Burda P, Aebi M. The dolichol pathway of N-linked glycosylation.Biochim Biophys Acta 1999;1426:239–57.

51. Kukuruzinska MA, Robbins PW. Protein glycosylation in yeast; tran-script heterogeneity of the ALG7 gene. Proc Natl Acad Sci U S A 1987;84:2145–9.

52. Helenius A, Aebi M. Intracellular functions of N-linked glycans. Science2001;291:2364–9.

53. Frank CG, Sanyal S, Rush JS,Waechter CJ, Menon AK. Does Rft1 flip anN-glycan lipid precursor? Nature 2008;454:E3–4.

54. Sanyal S,MenonAK. Specific transbilayer translocation of dolichol-linkedoligosaccharides by an endoplasmic reticulumflippase. ProcNatl AcadSciU S A 2009;106:767–72.

55. Sanyal S, Menon AK. Flipping lipids: why an' what's the reason for? ACSChem Biol 2009;4:895–909.

56. Rush JS, Gao N, Lehrman MA, Matveev S, Waechter CJ. Suppression ofRft1 expression does not impair the transbilayer movement of Man5-GlcNAc2-P-P-dolichol in sealed microsomes from yeast. J Biol Chem2009;284:19835–42.

57. Trombetta ES. The contribution of N-glycans and their processing in theendoplasmic reticulum to glycoprotein biosynthesis. Glycobiology2003;13:77R–91R.

58. Roth J. Protein N-glycosylation along the secretory pathway: relationshipto organelle topography and function, protein quality control, and cellinteractions. Chem Rev 2002;102:285–303.

59. Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides.Annu Rev Biochem 1985;54:631–64.

60. DunphyWG, Pfeffer SR,ClaryDO,Wattenberg BW,Glick BS, Rothman JE.Yeast andmammals utilize similar cytosolic components to drive proteintransport through the Golgi complex. Proc Natl Acad Sci U S A 1986;83:1622–26.

61. Rothenberg BE, Hayes BK, Toomre D, Manzi AE, Varki A. Biotinylateddiaminopyridine: an approach to tagging oligosaccharides and exploringtheir biology. Proc Natl Acad Sci U S A 1993;90:11939–43.

62. Ballou L, Hitzeman RA, Lewis MS, Ballou CE. Vanadate-resistant yeastmutants are defective in protein glycosylation. Proc Natl Acad Sci U S A1991;88:3209–12.

63. Herscovics A, Orlean P. Glycoprotein biosynthesis in yeast. FASEB J1993;7:540–50.

64. Dean N. Asparagine-linked glycosylation in the yeast Golgi. BiochimBiophys Acta 1999;1426:309–22.

65. Reason AJ, Dell A, Romero PA, Herscovics A. Specificity of the mannosyl-transferase which initiates outer chain formation in Saccharomyces cere-visiae. Glycobiology 1991;1:387–91.

66. Nakayama K-I, Nagasu T, Shimma Y, Kuromitsu J, Jigami Y. OCH1encodes a novel membrane bound mannosyltransferase: outer chainelongation of asparagine-linked oligosaccharides. EMBO J 1992;11:2511.

67. Liu B, Gong X, Chang S, Yang Y, Song M, Duan D, et al. Disruption of theOCH1 and MNN1 genes decrease N-glycosylation on glycoproteinexpressed in Kluyveromyces lactis. J Biotechnol 2009;143:95–102.

68. Nakanishi-Shindo Y, Nakayama K, Tanaka A, Toda Y, Jigami Y. Structureof theN-linkedoligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3mutants of Saccharomyces cerevisiae. J Biol Chem 1993;268:26338–45.

69. Nakayama K, Nakanishi-Shindo Y, Tanaka A, Haga-Toda Y, Jigami Y.Substrate specificity of alpha-1,6-mannosyltransferase that initiates N-linked mannose outer chain elongation in Saccharomyces cerevisiae.FEBS Lett 1997;412:547–50.

70. Lehle L, Eiden A, Lehnert K, Haselbeck A, Kopetzki E. Glycoproteinbiosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylationmutant allelic to och1 having a defect in the initiation of outer chainformation. FEBS Lett 1995;370:41–5.

71. Verostek MF, Trimble RB. Mannosyltransferase activities in membranesfrom various yeast strains. Glycobiology 1995;5:671–81.

72. Paulson JC, Colley KJ. Glycosyltransferases. Structure, localization,and control of cell type-specific glycosylation. J Biol Chem 1989;264:17615–8.

73. Ballou CE. Isolation, characterization, and properties of Saccharomycescerevisiae mnn mutants with nonconditional protein glycosylationdefects. In: David VG, editor. Methods in Enzymology. Cleveland, Ohio:Academic Press; 1990. p 440–70.

74. Kanik-Ennulat C, Montalvo E, Neff N. Sodium orthovanadate-resistantmutants of saccharomyces cerevisiae show defects in golgi-mediatedprotein glycosylation, sporulation and detergent resistance. Genetics1995;140:933–43.

75. Dean N, Poster JB. Molecular and phenotypic analysis of the S. cerevisiaeMNN10 gene identifies a family of related glycosyltransferases. Glyco-biology 1996;6:73–81.

76. Chapman RE, Munro S. The functioning of the yeast Golgiapparatus requires an ER protein encoded by ANP1, a memberof a new family of genes affecting the secretory pathway. EMBO J1994;13:4896–907.

77. Ballou L, Alvarado E, Tsai PK, Dell A, Ballou CE. Protein glycosylationdefects in the Saccharomyces cerevisiae mnn7 mutant class. Support forthe stop signal proposed for regulation of outer chain elongation. J BiolChem 1989;264:11857–64.

78. Gopal PK, Ballou CE. Regulation of the protein glycosylation pathway inyeast: structural control ofN-linked oligosaccharide elongation. ProcNatlAcad Sci U S A 1987;84:8824–8.

79. Ballou L, Hernandez LM, Alvarado E, Ballou CE. Revision of the oligo-saccharide structures of yeast carboxypeptidase Y. ProcNatl Acad Sci U S A1990;87:3368–72.

80. Hernandez LM, Ballou L, Alvarado E, Gillece-Castro BL, Burlingame AL,Ballou CE. A new Saccharomyces cerevisiae mnn mutant N-linked oligo-saccharide structure. J Biol Chem 1989;264:11849–56.

81. Munro S.What can yeast tell us about N-linked glycosylation in the Golgiapparatus? FEBS Lett 2001;498:223–7.

82. Yip CL, Welch SK, Klebl F, Gilbert T, Seidel P, Grant FJ, et al. Cloning andanalysis of the Saccharomyces cerevisiaeMNN9 and MNN1 genes requiredfor complex glycosylation of secreted proteins. Proc Natl Acad Sci U S A1994;91:2723–7.

83. Lussier M, Sdicu AM, Bussey H. The KTR andMNN1mannosyltransferasefamilies of Saccharomyces cerevisiae. Biochim Biophys Acta 1999;1426:323–34.

84. Wang XH, Nakayama K, Shimma Y, Tanaka A, Jigami Y. MNN6, amember of the KRE2/MNT1 family, is the gene for mannosylpho-sphate transfer in Saccharomyces cerevisiae. J Biol Chem 1997;272:18117–24.

85. Nakamura S, Takasaki H, Kobayashi K, Kato A. Hyperglycosylation of henegg white lysozyme in yeast. J Biol Chem 1993;268:12706–12.

86. Takashima S. Characterization of mouse sialyltransferase genes: theirevolution and diversity. Biosci Biotechnol Biochem 2008;72:1155–67.

87. Koles K, Irvine KD, Panin VM. Functional characterization of Drosophilasialyltransferase. J Biol Chem 2004;279:4346–57.

88. Khan W, Ashfaq UA, Aslam S, Saif S, Aslam T, Tusleem K, et al.Anticancer screening of medicinal plant phytochemicals againstCyclin-Dependent Kinase-2 (CDK2): an in-silico approach. Adv LifeSci 2017;4:113–9.

89. Hassan SA, Akhlaq F, Tayyab M, Awan AR, Firyal S, Khan WA, et al.Glyphosate: cancerous or not? Perspectives from both ends of the debate.Adv Life Sci 2017;4:108–12.

90. Berninsone PM,HirschbergCB.Nucleotide sugar transporters of theGolgiapparatus. Curr Opin Struct Biol 2000;10:542–7.

91. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, HenrissatB. TheCarbohydrate-Active EnZymes database (CAZy): an expert resourcefor Glycogenomics. Nucleic Acids Res 2009;37:D233–8.

92. Rhee SW, Starr T, Forsten-Williams K, Storrie B. The steady-state distri-bution of glycosyltransferases between the Golgi apparatus and theendoplasmic reticulum is approximately 90:10. Traffic 2005;6:978–90.

Nadeem et al.





93. Peanne R, Vanbeselaere J, Vicogne D, Mir AM, Biot C, Matthijs G, et al.Assessing ER and Golgi N-glycosylation process using metabolic labelingin mammalian cultured cells. Methods Cell Biol 2013;118:157–76.

94. Stanley P. Golgi glycosylation. Cold Spring Harb Perspect Biol 2011;3:a005199.

95. Oriol R, Mollicone R, Cailleau A, Balanzino L, Breton C. Divergentevolution of fucosyltransferase genes from vertebrates, invertebrates, andbacteria. Glycobiology 1999;9:323–34.

96. SchachterH. The joys of HexNAc. The synthesis and function ofN- andO-glycan branches. Glycoconj J 2000;17:465–83.

97. Liu C-P, Tsai T-I, Cheng T, Shivatare VS, Wu C-Y, Wu C-Y, et al. Gly-coengineering of antibody (Herceptin) through yeast expression and invitro enzymatic glycosylation. Proc Natl Acad Sci U S A 2018;115:720–5.

98. Nielsen J. Production of biopharmaceutical proteins by yeast: advancesthrough metabolic engineering. Bioengineered 2013;4:207–11.

99. Ko E, Kim M, Park Y, Ahn YJ. Heterologous expression of the carrotHsp17.7 gene increased growth, cell viability, and protein solubility intransformed yeast (Saccharomyces cerevisiae) under heat, cold, acid, andosmotic stress conditions. Curr Microbiol 2017;74:952–60.

100. Boutros C, Tarhini A, Routier E, Lambotte O, Ladurie FL, Carbonnel F,et al. Safety profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and incombination. Nat Rev Clin Oncol 2016;13:473–86.

101. ValsecchiME. Combined nivolumab and ipilimumab ormonotherapy inuntreated melanoma. N Engl J Med 2015;373:1270.

102. Philips GK, Atkins M. Therapeutic uses of anti-PD-1 and anti-PD-L1antibodies. Int Immunol 2015;27:39–46.

103. Pardoll DM. The blockade of immune checkpoints in cancer immuno-therapy. Nat Rev Cancer 2012;12:252–64.

104. Legat A, Speiser DE, Pircher H, Zehn D, Fuertes Marraco SA. Inhibitoryreceptor expression depends more dominantly on differentiation andactivation than "Exhaustion" of human CD8T cells. Front Immunol2013;4:455.

105. Cao J, Perez-Pinera P, Lowenhaupt K, Wu M-R, Purcell O, de la Fuente-Nunez C, et al. Versatile and on-demand biologics co-production in yeast.Nat Commun 2018;9:77.

106. Lussier M, Sdicu AM, Ketela T, Bussey H. Localization and targeting of theSaccharomyces cerevisiae Kre2p/Mnt1p alpha 1,2-mannosyltransferase to amedial-Golgi compartment. J Cell Biol 1995;131:913–27.

107. Chen X, Zhang W, Wang J, Fang J, Wang PG. Production of alpha-galactosyl epitopes via combined use of two recombinant whole cellsharboring UDP-galactose 4-epimerase and alpha-1,3-galactosyltransfer-ase. Biotechnol Prog 2000;16:595–9.

108. Bobrowicz P, Davidson RC, Li H, Potgieter TI, Nett JH, Hamilton SR, et al.Engineering of an artificial glycosylation pathway blocked in core oligo-saccharide assembly in the yeast Pichia pastoris: production of complexhumanized glycoproteins with terminal galactose. Glycobiology 2004;14:757–66.

109. Hamilton SR, Bobrowicz P, Bobrowicz B, Davidson RC, Li H, Mitchell T,et al. Production of complex human glycoproteins in yeast. Science2003;301:1244–6.

110. VTU Technology. VTU Technology and RCT Announce Partnership [2015Apr 3]. Available from: http://www.vtu-technology.com/VTU-Technology-and-RCT-Announce-Partnership/en/2025.

111. Beck A, Cochet O, Wurch T. GlycoFi's technology to control the glyco-sylation of recombinant therapeutic proteins. Expert Opin Drug Discov2010;5:95–111.

112. Nett JH, Gerngross TU. Cloning and disruption of the PpURA5 gene andconstruction of a set of integration vectors for the stable genetic modi-fication of Pichia pastoris. Yeast 2003;20:1279–90.

113. Nett JH, Hodel N, Rausch S, Wildt S. Cloning and disruption of the PichiapastorisARG1,ARG2,ARG3,HIS1,HIS2,HIS5,HIS6 genes and their use asauxotrophic markers. Yeast 2005;22:295–304.

114. Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, et al.Optimization of humanized IgGs in glycoengineered Pichia pastoris. NatBiotechnol 2006;24:210–5.

115. Beck A, Wagner-Rousset E, Ayoub D, Van Dorsselaer A, Sanglier-Cianf�erani S. Characterization of therapeutic antibodies and relatedproducts. Anal Chem 2012;85:715–36.

116. Hamilton SR, Davidson RC, Sethuraman N, Nett JH, Jiang Y, Rios S, et al.Humanization of yeast to produce complex terminally sialylated glyco-proteins. Science 2006;313:1441–3.

117. Walsh G, Jefferis R. Post-translational modifications in the context oftherapeutic proteins. Nat Biotechnol 2006;24:1241–52.

118. Takeuchi M, Kobata A. Structures and functional roles of the sugar chainsof human erythropoietins. Glycobiology 1991;1:337–46.

119. Hamilton SR, Gerngross TU. Glycosylation engineering in yeast: theadvent of fully humanized yeast. Curr Opin Biotechnol 2007;18:387–92.

120. Dumont J, Euwart D, Mei B, Estes S, Kshirsagar R. Human cell lines forbiopharmaceutical manufacturing: history, status, and future perspec-tives. Crit Rev Biotechnol 2016;36:1110–22.

121. Kunert R, Reinhart D. Advances in recombinant antibodymanufacturing.Appl Microbiol Biotechnol 2016;100:3451–61.

122. Quartino AL, Li H, Jin JY, Wada DR, Benyunes MC, McNally V, et al.Pharmacokinetic and exposure–response analyses of pertuzumab incombination with trastuzumab and docetaxel during neoadjuvant treat-ment of HER2þ early breast cancer. Cancer Chemother Pharmacol2017;79:353–61.

123. Krejcik J, Casneuf T, Nijhof IS, Verbist B, Bald J, Plesner T, et al. Dar-atumumab depletes CD38(þ) immune regulatory cells, promotes T-cellexpansion, and skews T-cell repertoire in multiple myeloma. Blood2016;128:384–94.

124. van Rhee F, Rothman M, Ho KF, Fleming S, Wong RS, Fossa�A, et al.

Patient-reported outcomes for multicentric Castleman's disease in arandomized, placebo-controlled study of siltuximab. Patient 2015;8:207–16.

125. Zimran A, Pastores GM, Tylki-Szymanska A, Hughes DA, Elstein D,Mardach R, et al. Safety and efficacy of velaglucerase alfa in Gaucherdisease type 1 patients previously treated with imiglucerase. Am J Hema-tol 2013;88:172–8.

126. Estes S, Melville M. Mammalian cell line developments in speed andefficiency. Adv Biochem Eng Biotechnol 2014;139:11–33.

127. Bandaranayake AD, Almo SC. Recent advances in mammalian proteinproduction. FEBS Lett 2014;588:253–60.

128. Almagro JC,Daniels-Wells TR, Perez-Tapia SM, PenichetML. Progress andchallenges in the design and clinical development of antibodies for cancertherapy. Front Immunol 2017;8:1751.

129. Wang Y, Sanchez L, Siegel DS,WangML. Elotuzumab for the treatment ofmultiple myeloma. J Hematol Oncol 2016;9:55.

130. Ma J, Li Q, Yu Z, Cao Z, Liu S, Chen L, et al. Immunotherapystrategies against multiple myeloma. Technol Cancer Res Treat2017;16:717–26.

131. Tran HC, Wan Z, Sheard MA, Sun J, Jackson JR, Malvar J, et al. TGFbR1blockade with galunisertib (LY2157299) enhances anti-neuroblastomaactivity of anti-GD2 antibody dinutuximab (ch14.18) with natural killercells. Clin Cancer Res 2017;23:804–13.

132. Flinn IW, Ruppert AS,HarwinW,WaterhouseD,Papish S, Jones JA, et al. APhase II study of two dose levels of ofatumumab induction followed bymaintenance therapy in symptomatic, previously untreated chronic lym-phocytic leukemia (CLL). Am J Hematol 2016;91:1020–5.

133. Rademacher TW, Homans SW, Parekh RB, Dwek RA. Immunoglobulin Gas a glycoprotein. Biochem Soc Symp 1986;51:131–48.

134. Carter P. Improving the efficacy of antibody-based cancer therapies. NatRev Cancer 2001;1:118–29.

135. Kanda Y, Yamada T, Mori K, Okazaki A, Inoue M, Kitajima-Miyama K,et al. Comparison of biological activity among nonfucosylated ther-apeutic IgG1 antibodies with three different N-linked Fc oligosacchar-ides: the high-mannose, hybrid, and complex types. Glycobiology2007;17:104–18.

136. NiwaR, Shoji-HosakaE, SakuradaM, ShinkawaT,UchidaK,NakamuraK,et al. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 withenhanced antibody-dependent cellular cytotoxicity shows potent thera-peutic activity to T-cell leukemia and lymphoma. Cancer Res 2004;64:2127–33.

137. Niwa R,Hatanaka S, Shoji-Hosaka E, SakuradaM, Kobayashi Y, Uehara A,et al. Enhancement of the antibody-dependent cellular cytotoxicity oflow-fucose IgG1 Is independent of FcgammaRIIIa functional polymor-phism. Clin Cancer Res 2004;10:6248–55.

138. Niwa R, Sakurada M, Kobayashi Y, Uehara A, Matsushima K, Ueda R,et al. Enhanced natural killer cell binding and activation by low-fucoseIgG1 antibody results in potent antibody-dependent cellular cytotox-icity induction at lower antigen density. Clin Cancer Res 2005;11:2327–36.





http://www.vtu-technology.com/VTU-Technology-and-RCT-Announce-Partnership/en/2025http://www.vtu-technology.com/VTU-Technology-and-RCT-Announce-Partnership/en/2025http://cancerres.aacrjournals.org/

139. Niwa R, Natsume A, Uehara A, Wakitani M, Iida S, Uchida K, et al. IgGsubclass-independent improvement of antibody-dependent cellular cyto-toxicity by fucose removal fromAsn297-linked oligosaccharides. J Immu-nol Methods 2005;306:151–60.

140. Suzuki E, Niwa R, Saji S, MutaM, Hirose M, Iida S, et al. A nonfucosylatedanti-HER2 antibody augments antibody-dependent cellular cytotoxicityin breast cancer patients. Clin Cancer Res 2007;13:1875–82.

141. Miyoshi E, Noda K, Yamaguchi Y, Inoue S, Ikeda Y, Wang W, et al. Thealpha1-6-fucosyltransferase gene and its biological significance. BiochimBiophys Acta 1999;1473:9–20.

142. Tonetti M, Sturla L, Bisso A, Benatti U, De Flora A. Synthesis of GDP-L-fucose by the human FX protein. J Biol Chem 1996;271:27274–9.

143. Becker DJ, Lowe JB. Fucose: biosynthesis and biological function inmammals. Glycobiology 2003;13:41R–53R.

144. Mori K, Kuni-Kamochi R, Yamane-Ohnuki N, Wakitani M, Yamano K,Imai H, et al. Engineering Chinese hamster ovary cells to maximizeeffector function of produced antibodies using FUT8 siRNA. BiotechnolBioeng 2004;88:901–8.

145. Imai-Nishiya H, Mori K, Inoue M, Wakitani M, Iida S, Shitara K, et al.Double knockdown of a1,6-fucosyltransferase (FUT8) and GDP-man-nose 4,6-dehydratase (GMD) in antibody-producing cells: a new strategyfor generating fully non-fucosylated therapeutic antibodies withenhanced ADCC. BMC Biotech 2007;7:84–84.

146. Zhang F, Qi X, Wang X, Wei D, Wu J, Feng L, et al. Structural basis of thetherapeutic anti-PD-L1 antibody atezolizumab. Oncotarget 2017;8:90215–24.

147. Goletz S, Danielczyk A, Stahn R, Karsten U, Stoeckl L, Loeffler A, et al.GlycoOptimization for fully human and largely improved biopharma-ceutical antibodies and proteins. Glyco-Bioinformatics 2010.

148. Noguchi A, Mukuria CJ, Suzuki E, Naiki M. Immunogenicity of N-glycolylneuraminic acid-containing carbohydrate chains of recombinanthuman erythropoietin expressed in Chinese hamster ovary cells. J Bio-chem 1995;117:59–62.

149. Chung CH, Mirakhur B, Chan E, Le Q-T, Berlin J, Morse M, et al.Cetuximab-induced anaphylaxis and IgE specific for galactose-a-1,3-galactose. N Engl J Med 2008;358:1109–17.

150. Wang Q, Yin B, Chung CY, Betenbaugh MJ. Glycoengineering ofCHO cells to improve product quality. Methods Mol Biol 2017;1603:25–44.

151. Yang S, Zhang L, Thomas S, Hu Y, Li S, Cipollo J, et al. Modification ofsialic acids on solid phase: accurate characterization of protein sialylation.Anal Chem 2017;89:6330–5.

152. Lee FW, Elias CB, Todd P, Kompala DS. Engineering Chinese hamsterovary (CHO) cells to achieve an inverse growth - associated production ofa foreign protein, beta-galactosidase. Cytotechnology 1998;28:73–80.

153. Lepenies B, Seeberger PH. Simply better glycoproteins. Nat Biotechnol2014;32:443–5.

154. Meuris L, Santens F, Elson G, Festjens N, Boone M, Dos Santos A, et al.GlycoDelete engineering of mammalian cells simplifies N-glycosylationof recombinant proteins. Nat Biotechnol 2014;32:485–9.

155. Reeves PJ, CallewaertN, Contreras R, KhoranaHG. Structure and functionin rhodopsin: high-level expression of rhodopsin with restricted andhomogeneous N-glycosylation by a tetracycline-inducible N-acetylglu-cosaminyltransferase I-negative HEK293S stable mammalian cell line.Proc Natl Acad Sci U S A 2002;99:13419–24.

156. Verstraete K, Vandriessche G, Januar M, Elegheert J, ShkumatovAV, Desfosses A, et al. Structural insights into the extracellularassembly of the hematopoietic Flt3 signaling complex. Blood 2011;118:60–8.

157. Velasquez EV, RiosM,OrtizME, Lizama C, Nunez E, AbramovichD, et al.Concanavalin-A induces granulosa cell death and inhibits FSH-mediatedfollicular growth and ovarian maturation in female rats. Endocrinology2013;154:1885–96.

158. Lee F, Yokota T, Otsuka T, Gemmell L, Larson N, Luh J, et al. Isolation ofcDNA for a human granulocyte-macrophage colony-stimulating factor byfunctional expression in mammalian cells. Proc Natl Acad Sci U S A1985;82:4360–4.

159. Lux A, Yu X, Scanlan CN,Nimmerjahn F. Impact of immune complex sizeand glycosylation on IgG binding to human FcgammaRs. J Immunol2013;190:4315–23.

160. Tradtrantip L, Ratelade J, Zhang H, Verkman AS. Enzymatic deglycosyla-tion converts pathogenic neuromyelitis optica anti-aquaporin-4 immu-noglobulin G into therapeutic antibody. Ann Neurol 2013;73:77–85.

161. Nandakumar KS, Collin M, Happonen KE, Croxford AM, Lundstrom SL,Zubarev RA, et al. Dominant suppression of inflammation by glycan-hydrolyzed IgG. Proc Natl Acad Sci U S A 2013;110:10252–7.

162. Allhorn M, Collin M. Sugar-free antibodies–the bacterial solution toautoimmunity? Ann N Y Acad Sci 2009;1173:664–9.

163. Barkdull S, Brownell I. PD-L1 blockade with avelumab: a new paradigmfor treating Merkel cell carcinoma. Cancer Biol Ther 2017;18:937–9.

164. Chism DD. Urothelial carcinoma of the bladder and the rise of immu-notherapy. J Natl Compr Canc Netw 2017;15:1277–84.

165. Jabbour E, Ravandi F, Kebriaei P, Huang X, Short NJ, Thomas D, et al.Salvage chemoimmunotherapy with inotuzumab ozogamicin combinedwith mini-hyper-CVD for patients with relapsed or refractory philadel-phia chromosome-negative acute lymphoblastic leukemia: a phase 2clinical trial. JAMA Oncol 2018;4:230–4.

166. Abdel-Rahman O. Smoking and EGFR status may predict outcomes ofadvanced NSCLC treated with PD-(L)1 inhibitors beyond first line; ameta-analysis. Clin Respir J 2017. doi: 10.1111/crj.12742.

167. Bekoz H, Karadurmus N, Paydas S, Turker A, Toptas T, Firatli Tuglular T,et al. Nivolumab for relapsed or refractory Hodgkin lymphoma: real-lifeexperience. Ann Oncol 2017;28:2496–502.

168. Tikhonova IA, Jones-Hughes T, Dunham J, Warren FC, Robinson S,Stephens P, et al. Olaratumab in combination with doxorubicin for thetreatment of advanced soft tissue sarcoma: an evidence review groupperspective of a National Institute for Health and Care Excellence SingleTechnology Appraisal. Pharmacoeconomics 2018;36:39–49.

169. Haque S, Yellu M, Randhawa J, Hashemi-Sadraei N. Profile of pembro-lizumab in the treatment of head and neck squamous cell carcinoma:design development and place in therapy. Drug Des Dev Ther2017;11:2537–49.

170. Prat A, Navarro A, Pare L, Reguart N, Galvan P, Pascual T, et al. Immune-related gene expression profiling after PD-1 blockade in non-small celllung carcinoma, head and neck squamous cell carcinoma, and melano-ma. Cancer Res 2017;77:3540–50.

171. Zhang T,Wang S, Lin T, Xie J, Zhao L, Liang Z, et al. Systematic review andmeta-analysis of the efficacy and safety of novel monoclonal antibodiesfor treatment of relapsed/refractory multiple myeloma. Oncotarget2017;8:34001–17.

172. Erbe AK, Wang W, Carmichael L, Kim K, Mendonca EA, Song Y, et al.Neuroblastoma patients' KIR and KIR-ligand genotypes influence clinicaloutcome for dinutuximab-based immunotherapy: a report from theChildren's Oncology Group. Clin Cancer Res 2018;24:189–96.

173. Terzic T, Cordeau M, Herblot S, Teira P, Cournoyer S, Beaunoyer M, et al.Expression of disialoganglioside (GD2) in neuroblastic tumors: a prog-nostic value for patients treated with anti-GD2 immunotherapy. PediatrDev Pathol 2017. doi: 10.1177/1093526617723972.

174. Liu J, Yang H, Liang X, Wang Y, Hou J, Liu Y, et al. Meta-analysis of theefficacy of treatments for newly diagnosed and relapsed/refractory mul-tiple myeloma with del(17p). Oncotarget 2017;8:62435–44.

175. Pazina T, James AM, MacFarlane AWt, Bezman NA, Henning KA, Bee C,et al. The anti-SLAMF7 antibody elotuzumabmediates NK cell activationthroughbothCD16-dependent and -independentmechanisms.Oncoim-munology 2017;6:e1339853.

176. Brinkmeyer JK, Moore DC. Necitumumab for the treatment of squamouscell non-small cell lung cancer. J Oncol Pharm Pract 2018;24:37–41.

177. Motoo Y. Ramucirumab plus paclitaxel: a possible new chemotherapyregimen for neuroendocrine carcinoma of the stomach. Intern Med2018;57:631–2.

178. Muro K, Cho JY, Bodoky G, Goswami C, Chao Y, Dos Santos LV, et al. Agedoes not influence efficacy of ramucirumab in advanced gastric cancer:subgroup analyses of REGARD and RAINBOW. J Gastroenterol Hepatol2018;33:814–24.


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