Glycosylation of Recombinant Anticancer Therapeutics in Different Expression Systems with Emerging Technologies

Published OnlineFirst May 22, 2018; DOI: 10.1158/0008-5472.CAN-18-0032 Cancer Review Research

Glycosylation of Recombinant Anticancer Therapeutics in Different Expression Systems with Emerging Technologies Tariq 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 major system to obtain structurally and functionally identical glycans, role in recombinant anticancer therapeutic proteins, as most of as in humans. In many expression systems, the N-glycosylation the approved recombinant therapeutics are glycoproteins. The pathway remains conserved in the endoplasmic reticulum, constant amino acid sequence of therapeutics determines the but divergence is observed when the protein enters the Golgi enzymatic activity, while the presence of glycans influences complex. Hence, in recent decades, numerous approaches their pharmacokinetics, solubility, distribution, serum half-life, have been adopted to engineer the Golgi's N-glycosylation effector function, and binding to receptors. Glycoproteins pathway to attain human-like glycans. Several researchers have expressed in different expression systems acquire their own tried to engineer the N-glycosylation pathway of expression oligosaccharides, which increases the protein diversity. The systems. In this review, we examine the glycosylation pattern heterogeneity of glycans creates hurdles in downstream proces- in various expression systems, along with emerging technologies sing, ultimately leading to variable anticancer therapeutic effi- for glycosylation engineering of anticancer therapeutic drugs. cacy. Therefore, glycoproteins require an appropriate expression Cancer Res; 78(11); 1–12. 2018 AACR.

Introduction ket value of protein-based drugs is growing, with a compounded annual growth rate of 16% compared with the pharmaceutical Cancer is the second leading cause of death in humans, devour- market growth rate of 8% (9). Among the total approved bio- ing the lives of 8.8 million people in 2015 (1, 2). In 2025, 19.3 pharmaceuticals, almost 70% are glycoproteins, which contain million new cases are predicted (3). This disease is characterized carbohydrate moieties gained as a posttranslational modification by abnormal and uncontrolled growth of cells, which have the in the process of glycosylation (10–13). This glycosylation diver- potential to invade other parts of the body through metastasis sifies the class of biopharmaceuticals. Many functions of antican- (4, 5). Currently, most common cancer treatments include cer glycoproteins are associated with glycan attachments, such as radiotherapy, surgery, and chemotherapy. With the advancement solubility, pharmacodistribution, pharmacokinetics, proper of technologies, efforts are being made in clinical treatment to structural folding, binding to receptors, and serum half-life (14). identify effective state-of-the-art therapies to replace conventional The most significant anticancer therapeutic recombinant pro- methods (6, 7). teins are mAbs, which are glycosylated in their Fc region (15). Recent advances have paved the way for the development of Alteration of the composition and structure of glycans causes recombinant anticancer therapeutics through engineered cell conformational changes in the Fc domain of antibodies, affecting lines. As anticancerous agents, these drugs improve the delivery their binding affinity to Fcg receptors (16, 17). This process leads of immune cells to tumor tissues, altering the tumor microenvi- to a change in immune effector functions, including complement- ronment, enhancing antigen priming, and facilitating effector cell dependent cytotoxicity, antibody-dependent cell-mediated cyto- activation and maturation (6, 7). Production of anticancer ther- toxicity (ADCC), and antibody-dependent cell-mediated phago- apeutic proteins as a class of drugs is dominating the drug cytosis (18). Deglycosylation of antibodies reduces their binding industry, partly because of the high demand and partly because affinity and hence their effector functions (19, 20). Changes in the of advancements in recombinant DNA technology (8). The mar- glycoforms of therapeutic mAbs or Fc-fusion proteins can impact the pharmacokinetics of proteins; for example, the negative impact of hypermannosylation on pharmacokinetics can trigger 1Center of Excellence in Molecular Biology, University of the Punjab, Lahore, the C-type lectin clearance mechanism (15, 18, 21). In many cases, Pakistan. 2Institute of Molecular Biology and Biotechnology, University of the terminal sugars in the glycans can affect the pharmacokinetics 3 Lahore, Lahore, Pakistan. Department of Microbiology, Quaid-I-Azam Univer- of an antibody due to glycan binding to receptors on tissues, sity, Islamabad, Pakistan. ultimately leading to its removal from circulation. The major Corresponding Authors: Qurban Ali, Center of Excellence in Molecular Biology, glycan receptors that remove glycoproteins are the mannose University of the Punjab, Lahore 57300, Pakistan. Phone: 321-962-1929; E-mail: receptor and the asialoglycoprotein receptor (22, 23). As both [email protected]; and Tariq Nadeem, [email protected] these receptors are abundantly expressed in the liver, it is likely doi: 10.1158/0008-5472.CAN-18-0032 that glycoproteins with terminal mannose or galactose residues 2018 American Association for Cancer Research. will be distributed predominantly in the liver and be catabolized

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there, as shown by Wright and colleagues in the case of an IgG with their effector functions. This article reviews N-glycosylation occur- terminal mannose or galactose residues (24). ring in different expression systems, and we have summarized the Development of recombinant anticancer therapeutics has various strategies adopted in different glycosylation engineering made substantial progress for the treatment of various solid and technologies. hematologic tumors over the past decade (25–27). Naked mAbs are most commonly used to treat cancer. Rituximab was the first N-glycosylation in the Endoplasmic fi recombinant mAb approved by FDA in 1997 and nds its ther- Reticulum apeutic applications in variety of hematologic cancers including lymphocytic leukemia and non-Hodgkin lymphoma. It is an Eukaryotic N-glycosylation occurs in two organelles, the endo- anti-CD20 humanized recombinant drug that plays a pivotal role plasmic reticulum (ER) and the Golgi complex. Dolichol-linked in B-cell malignancies (28–30). On the other hand, trastuzumab glycan precursor formation and transfer to a nascent protein with is one of the mAbs used for the treatment of solid tumor that is a little bit of processing occurs in the ER, while complete matu- þ capable of ADCC through interactions with Fcg/R immune cell ration and processing of N-linked glycans occurs in the Golgi subsets. It has transformed the treatment of HER-2–positive breast apparatus. N-glycosylation occurs at asparagine residue in the cancer (31–33). Tumor targeted recombinant mAbs can also be consensus amino acid sequence (Asn-X-Ser/Thr), where X can be conjugated to other forms of anticancer therapy that enhances any amino acid, but not proline. The process is initially started and their efficacy by lessening the systemic toxicities to normal cells. processed in the ER, where oligosaccharide transferase (OT) There are three types conjugated mAbs: radiolabeled that are catalyzes the transfer of glycan onto the nascent protein (48). attached to radionuclide moieties, chemolabeled that are linked Secretory proteins containing signal peptides are directed by to antineoplastic drugs, and immunotoxin mAbs that are associ- signal recognition particles across the membrane into the lumen ated with bacterial and plant toxins (34–36). Revolution in of the ER, followed by its movement into the OT-mediated recombinant DNA technology has facilitated the progress toward glycosylation machinery. Glycosylation is not dependent on more specific and less toxic anticancer therapy (29). protein folding or tertiary structure. However, some evidence has In eukaryotic organisms, N-glycosylation is the most prevalent shown that secondary structures on both sides of the Asn con- type of glycosylation, in which a preassembled oligosaccharide is sensus sequences may help in this enzymatic reaction (48). The transferred onto asparagine (Asn) in the consensus sequence of whole process of glycosylation is completed in the ER and Golgi the nascent protein. This oligosaccharide processing and matu- bodies. Glycoproteins processed in the ER usually show homol- ration occurs regardless of the protein template (12, 37–40). ogy and remain conserved in higher eukaryotes and yeast (49). Hence, cancer glycoproteins expressed in different expression Tetradecasaccharide (Glc3Man9GlcNAc2b1) attached to Asn- systems acquire glycans depending on their own glycosylation amide groups is derived from the dolichol pathway (50). The machinery. Glycoproteins expressed in yeast show hypermanno- synthesis of tetradecasaccharide starts on the cytosolic face of the sylated glycans, which compromise their therapeutic efficacy ER by transferring GlcNAc onto the membrane-anchored Dol-P, (41–45). Similarly, glycoproteins expressed in plant cells acquire yielding Dol-PP-GlcNAc in a Alg7-catalyzed reaction (51). In xylose residues on their glycans, which show similar results as a further steps, mannose residues are attached by mannosyltrans- therapeutic agent (46, 47). Until recently, mammalian cells had ferase using the substrate GDP-Man. The first five mannoses are a prominent role in glycoprotein production, but alterations in attached on the cytosolic side of the ER. Glycan (32) is then their glycosylation pathway to produce human-like glycans are flipped onto the luminal side by the membrane spinning flippase needed. Therefore, glycans, being an important protein-quality Rft1p (52). Recently, biochemical studies have revealed that attribute, required a humanized glycosylation machinery for flippase (Rft1p) may not be required for this process (53–56). their processing. In the remaining steps, four mannosyltransferases and three The rapid growth of glycoproteins and the associated financial glycosyltransferases catalyze the reaction using Dol-P-Man and interest has compelled many researchers and companies to ana- Dol-P-Glc as substrates, adding mannose and glucose residues, lyze glycans. In recent years, several engineering technologies have respectively (48). After the formation of tetradecasaccharide is been introduced that successfully engineered the glycosylation completed, OT transfers it to the Asn residue of the nascent pathways of different expression systems (Table 1). The common protein. Attachment of tetradecasaccharide is followed by trim- goal of all these emerging technologies is to attach homogeneous ming of two glucose residues catalyzed by glucosidase I and and human-friendly glycans on therapeutic proteins to enhance glucosidase 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 163 Durvalumab Urothelial carcinoma May 2017 164 Inotuzumab ozogamicin B-cell precursor acute lymphoblastic leukemia August 2017 165 Atezolizumab Urothelial carcinoma and metastatic non-small cell lung cancer May 2016 146, 166 Nivolumab Classical hodgkin lymphoma May 2016 167 Olaratumab Soft tissue sarcoma October 2016 168 Pembrolizumab Head and neck squamous cell cancer August 2016 169, 170 Daratumumab Multiple myeloma November 2015 171 Dinutuximab Pediatrics with neuroblastoma March 2015 172, 173 Elotuzumab Mutiple myeloma November 2015 174, 175 Necitumumab Metastatic squamous non-small cell lung cancer November 2015 176 Ramucirumab Gastric cancer April 2014 177, 178

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cycle for proper folding (38, 52, 57, 58). In the last step, gluco- N-glycosylation in the Golgi Complex sidase II removes the third glucose, allowing the protein to enter Yeast Golgi the Golgi complex, where species- and cell-type–speciﬁc glyco- The protein glycans formed in the ER are well conserved in sylation and the remaining glycosylation process occur. In differ- different eukaryotes, which are progressively changed by different ent expression systems, the N-glycosylation pathway diverges at glycosyltransferases residing in the Golgi complex (60). This this step (59). The entire process is described in Fig. 1. modiﬁcation is highly diverse among different organisms and

Cytoplasm

UDP UDP UDP UDP UDP UDP UDP Rft1p Alg7 Alg13/14 Alg1 Alg2? Alg2? Alg11 Alg11?

ER Lumen Glucose Mannose GlcNAc Dolichyl-pyrophosphate Alg3 Dol-P

Alg9 Dol-P

Dol-P Dol-P Dol-P Dol-P Dol-P OT Alg10 Alg8 Alg6 Alg9 Alg12

Oligosaccharyl transferase

Endoplasmic reticulum

Figure 1. Synthesis of precursor oligosaccharide on the membrane of endoplasmic reticulum and transfer on the nascent protein. Biosynthesis of oligosaccharide is catalyzed by glycosyltransferases encoded by different ALG loci. First synthesis starts on the cytoplasmic face of the endoplasmic reticulum, which is then ﬂipped into the lumen 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 The extent of glycosylation of glycoproteins expressed in yeast also (61). In the yeast Saccharomyces cerevisiae, N-linked glycan's outer depends on culture medium (85), culture conditions, availability chain is further extended in the Golgi complex with mannose of substrates, and the transportation rate through the ER and residues. The number of mannose residues can reach up to Golgi. The use of old culture compared with fresh culture for 200 with the linear backbone containing up to 50 mannose glycoprotein expression can affect the glycan, as observed in residues linked via a-1,6 linkages. Further branching occurs secreted exoglucanase (64). through a-1,2 and a-1,3 linkages, resulting in hypermannosylation (62, 63). Several monosylphosphates also attach to an outer Mammalian Golgi chain, giving a negative charge to the oligosaccharides (64). Approximately 250 glycosyltransferases transfer sugars in the Secretory and cell wall glycoproteins are mostly hypermannosy- Golgi from donor to acceptor glycans on proteins and lipids in lated, and the glycan may contribute up to 95% of its molecular mammals. Up to 20 glycosyltransferases (86) are involved in the weight. Some intracellular glycoproteins usually escape these transfer of sialyl sugar in mammalian Golgi. Drosophila has just fi – modi cations and remain intact with short glycans of 9 13 one glycosyltransferase (87–89), whereas no such glycosyltrans- mannose residues (Fig. 2; ref. 54). ferase has been found in yeast Golgi. This is the reason glycopro- The substrate Man8GlcNAc, which comes directly from the teins expressed in yeast lack sialyl moieties in their glycans. These ER, is used by a-1,6-mannosyltransferase, encoded by the OCH1 sugars are mainly produced in the cytoplasm and rarely in the – gene, to add a single mannose to initiate the outer chain (65 67). nucleus, that is, CMP-Sia. Then, they are transported into the No trimming of Man8GlcNAc occurs in yeast, unlike higher Golgi lumen by multitransmembrane transporter families eukaryotes, before outer chain initiation (64). There is no evi- (90, 91). Each glycosyltransferase is used at a specific step and, dence of oligosaccharides smaller than Man8GlcNAc for manno- hence, is localized in specific compartments, such as the cis-Golgi, sidases in the Golgi. Therefore, the preference of OCH1 is very medial-Golgi, trans-Golgi, or trans-Golgi network (92). Once a narrow (68, 69) and is mostly Man8GlcNAc in vivo as well as glycoprotein is exported into the Golgi, N-linked glycans undergo in vitro (70, 71). OCH1 still initiates outer chain formation, but if several processes. In these processes, glycosidases carry out the found to be correct, a-1,3-mannose attaches to an incomplete trimming, while glycosyltransferases transfer sugar moieties onto core oligosaccharide from the ER in vivo but shows reduced activity the glycan (93). After a sugar is transported, a new intermediate on the same substrate in vitro (69, 71). substrate for another glycosyltransferase is created (94). Any sugar Glycan backbone outer chain elongating enzymes, mannosyl- containing a free hydroxyl group can be substituted in an inter- transferases, are divided into two gene families. They include the mediate glycan and, thus, many branch antennae are expected (95). VAN1, ANP1/MNN8, and MNN9 family and the MNN10 and Trimming of glycans occurs in the cis-Golgi, where mannosi- MNN11 family. These two families provide type II membrane dase I catalytically removes all a-1,2-linked mannose residues. At encoding proteins, which are unique of all the known Golgi this stage, three mannose residues are removed. N-acetylglucosa- glycosyltransferases in higher eukaryotes (72). Mutation in any minyltransferase I (GlcNAc-TI) adds GlcNAc to the man-a-1,3 of these genes can results in truncated oligosaccharide backbones arm of Man GlcNAc , which is a branched structure formed as a – 5 2 on glycoproteins (62, 73 76). Mnn9 uses the substrate catalyzed result of mannosidase I activity. Later, GlcNAc-TI converts high by OCH1. Mutant mnn9 allows the addition of only one a-1,6- mannose-type glycans into a hybrid and complex type by the linked mannose. This is followed by a-1,2-mannose, which is addition of GlcNAc. In the medial-Golgi, a-1,3- and a-1,6-linked believed to be a stop signal. As a consequence, the elongating mannose is removed by mannosidase II. GlcNAc is then added chain is terminated (77, 78), and then, it resembles the short onto a-1,6 mannose by GlcNAc-TII. In this way, a hybrid-type – glycan of some intracellular glycoproteins (79 81). Data of glycan is converted into a complex-type glycan. Many branches mnn9, mnn8, and mnn10 show that these enzymes function in that become biantenary, triantenary, tetra-antenary, and penta- a common pathway, where mnn9 acts before mnn8 and mnn10 antenary oligosaccharides can be generated by GlcNAc-TIV, (82). VAN1-mutant data revealed that this protein is involved in GlcNAc-TV, and/or GlcNAc-TVI. GlcNAc-TIII can prevent the internal a-1,2-mannose branching or backbone elongation. The activity of GlcNAc-TII, GlcNAc-TIV, GlcNAc-TV, and mannosidase VAN1 mutant provides mnn9-like glycosaccharides (62, 64). In II as it brings bisecting GlcNAc residue onto b-mannose of the core further steps, the core backbone is decorated with a-1,2-mannose, (93, 96). Fucosyltransferase can then add fucose residue to the a-1,3-mannose, and mannosylphosphate containing branches. very first GlcNAc directly attached to Asn in the polypeptide. The enzymes participating in this branching areMnn1p, which Fucose addition occurs in the medial-Golgi. In most of the cases, catalyzes the terminal a-1,3-mannose addition. Ktr2p, ktr1p, following the fucose addition, glycoprotein is shifted to the kre2p, and Yur1p are a-1,2-transfereases. Mnn6p is a mannosyltrans-Golgi for terminal glycosylation. Galactose and sialic acid phosphotransferase (83, 84). are attached to each N-glycan antenna. Galactose is usually added The last crucial step in glycosylation is chain termination. The by b-1,4 and b-1,3 galactosyltransferase. In the end, terminal sialic contributing factors involved in chain termination are not well acid is added to galactose by sialyltransferase. The most common understood. The distinguishing factors of hypermannosylation sialic acid in humans is NeuAc, which is added in an a-2,3, a-2,6, and hypomannosylation are still not known. Therefore, these two or a-2,8 linkage to galactose (93). processes cannot be differentiated yet (64). However, it has been suggested that a-1,2–linked mannose decides the fate of outer N-glycosylation Engineering Technologies chain elongation termination as a "stop signal." The oligosaccharides containing terminal a-1,2-linked mannose cannot be for Yeast used as a substrate by mannosyltransferase (78). However, the S. cerevisiae is a robust expression system for heterologous "stop signal" is not the only reason for chain termination, as some recombinant drugs production. Because of possibly higher titers, incomplete monosaccharides do not possess terminal mannose. low risk of human viral contamination and low scalable

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ER (Human, Yeast, Plants)

Man8BGIcNAc2

Golgi (Human) Golgi (Yeast) Golgi (Plants)

Man GIcNAc Man GIcNAc 5 2 9 2 Mns 1/2 GnT I 1, 2 MnTs 1, 2 MnTs

GIcNAcMan5GIcNAc2 GnT I

Mns II *

* Gm II GIcNAcMan3GIcNAc2

GnT II β-1, 4-Gal GnT II α-1, 2-Man α-1, 6-Man GIcNAc2Man3GIcNAc2 α-1, 3-Man XyIT GalT β-1, 4-Man FuT 11/12 β-1, N-GlcNAc β-1, 4-GlcNAc GaI2GlcNAc2Man3GIcNAc2 β-1, 2-GlcNAc GalT I ST α-2, 3-NANA/α-2, 6-NANA FuT 13 Present in S. cerevisiae but not in P. pastroris Xylose NANA GaI GlcNAc Man GIcNAc 2 2 2 3 2 Fucose

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. fermentation process, yeast-based protein production platform CTLA4 regulate T cells through negative feedback mechanism, is regarded as an alternative to mammalian expression system but they are upregulated at different stages of T-cell activation. (97–99). Mixture of two anticancer therapeutic proteins, anti- The human anticytotoxic T-lymphocyte–associated antigen 4 CTLA4 and anti-PD1, has been produced in yeast. These recom- antibody attaches to CTLA4 on the T-cell surface, preventing binant antibodies act as checkpoint inhibitors approved for CTLA4 from inhibiting T-cell activation, whereas the human management of advanced melanoma (100–102). PD1 and anti-programmed cell death 1 antibody binds to PD1, blocking

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tumor cells from shutting down T-cell activity (103, 104). Mixture domain of human b-1,4-galactosyltransferase I, catalyzesgalac- of these recombinant antibodies was produced by coculturing tose addition. For proper Golgi localization, the S. cerevisiae two Pichia pastoris strains such that each produced one of domain was included in the fusion protein Mnn2p (8) [this the mAbs under optimized culture conditions. Confirmation strategy was used for the first time for Escherichia coli (107) of correct structures and targets of the antibodies produced in and GlycoFi adapted it for P. pastoris (108)]. The resulting P. pastoris was affirmed through binding and competitive assays. modified strain GalGnM5 was able to produce a hybrid-type This reflects production of multiple recombinant anticancer GalGlcNAcMan5GlcNAc glycan. therapeutics in P. pastoris by integrating inducible pro- Furthermore, engineering of complex and hybrid-type glycans tein expression systems. Thus, we envision that this expression was carried out by introducing mannosidase II and GlcNActrans- system has the potential to reduce time, cost, number of strains, ferase II (GnT-II). First, the catalytic domain of mannosidase II and facilities required for anticancer therapeutics production from Drosophila melanogaster was fused with the S. cerevisiae (105). In this regard, various technologies have been introduced Mnn2P Golgi localization domain. Introduction of this fusion to humanized yeast expression system (Table 2). protein resulted in a GnM3 strain, which was able to remove terminal a-1,3 and a-1,6-linked mannose. Hence, the GnM3 GlycoSwitch technology strain modified its glycoproteins with a GlcNAcMan3GlcNAc2- Jacobs and colleagues have successfully engineered the type glycan. Transforming the fusion protein Mnn2DmMan-II N-glycosylation pathway of the yeast P. pastoris for the production into GalGnM5 strain resulted in a GalGnM3 strain, which could of humanized glycoproteins (10). In their strategy, they knocked modify N-glycan with a GalGlcNAcMan3GlcNAc2 glycan struc- out a gene involved in hypermannosylation and introduced ture. In the very last step for the addition of terminal galactose various mammalian pathway genes to attain human-like onto the biantennary complex type glycan, GnT-II was intro- N-glycan on recombinant glycoproteins. A total of five Glyco- duced. The catalytic domain of Rat GnT-II was fused with the Switch vectors were introduced, step by step, containing different S. cerevisiae Mnn2p N-terminal domain (109). The resulting selection markers. The protocol followed is not suitable for strain, Gal2Gn2M3, was capable of synthesizing Gal2GlcNAc2- resolving the issue of nonhuman O-linked glycosylation in Man3GlcNAc-type N-glycans. Three different types of proteins, P. pastoris (8). As described previously, the pattern of glycan mouse IL10, mouse GM-CSF, and mouse IL22, which have remains conserved at the ER level and diverges when it enters N-glycosylation sites that were expressed in each of these into the Golgi complex (59). To stop this hypermannosylation, strains, were produced as a result of engineering. N-linked the a-1,6-mannosyltransferase OCH1 gene was disrupted, as hyperglycosylation was successfully controlled using these it initiates the outer chain leading to hypermannosylated strains. It was also observed that the strain that was extensively branches. For this inactivation, the pGlycoSwitchM8 vector was engineered had increased glycan heterogeneity. This heteroge- used, which replaces the actual OCH1 with a nonactive fragment neity is believed to be caused by incomplete processing and by homologous recombination. The strain M8 produced as a hindrance created by endogenous monosyltransferases. This result of this inactivation was able to control hyperglycosylation at can be overcome by optimizing growth conditions and a the Man8GlcNAc glycan level. After this, HDEL-tagged a-1,2- culturing medium (8). Pichia GlycoSwitch has joined hands mannosidase, from Trichoderma reesei fungus, was introduced. in December 2014 with UTV Technologies, where they can use The resulting strain M5 successfully modified the glycan to Man5- VTU Technology yield-enhancing P. pastoris expression plat- GlcNAc, as the introduced gene had mostly removed all terminal form. UTV has the broadest toolbox and versatile technologies a-1,2–linked mannose residues (42). for expressing recombinant proteins in P. pastoris and has To convert N-glycan into a hybrid-type, GlcNAc transferase I already achieved the target of 22 g/L of secretory proteins. The (GnT-I) was introduced. For this purpose, the human GnT-I partnership of both technologies can ensure the better yield of catalytic domain was fused with the Kre2p N-terminus domain humanized anticancer glycoproteins (110). of S. cerevisiae (42). The Kre2p contributes to proper cis/medial- Golgi localization (106). The resulting strain, GnM5, was able to GlycoFi technology modify the glycan into GlcNAcMan5GlcNAc2. The next step in In an attempt to humanize the glycosylation pathway of yeast engineering was to add galactose to b-1,2-GlcNAc. For this, the for humanized glycoproteins, GlycoFi Inc. was founded by Pro- GnM5 strain was transformed with the pGlycoSwitchGalT vector. fessor Gerngross and Professor Hutchinson in 2000. In GlycoFi The vector had a tripartite fusion protein. The first part, UDP-Gal technology, a total of four genes of P. pastoris were knocked out, 4-epimerase, converts UDP-Glc into UDP-Gal, thus ensuring its and 14 genes were introduced. Consequently, the modified strains availability in the Golgi complex. The second part, the catalytic could 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, Transferrin Glycode (FR) GlycodExpress Saccharomyces cerevisiae (Yeast) Merck (US) GlycoFi Pichia pastoris (Yeast) EPO Glycotope GlycoExpress Human cell lines EGFR, HER2 GlycoDelete HEK GM-CSF, anti-CD20 siRNA mediated glycoengineering CHO (hamster) IgG1 Kyowa Hakko Kirin (JP) Lonza (UK) POTELLIGENT CHO (hamster) CCR4, CD98, GM2, IL5 Roche-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 growth factor; FR, France.

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complex N-glycans, similar to humans. The genes introduced necitumumab (EGFR antagonist) that are active in treatment mostly consisted of catalytic domains from mammalian origin of multiple myeloma, colorectal cancer, neuroblastoma, chronic and signal peptides from fungi for proper Golgi localization lymphocytic leukemia, and nonsmall cell lung cancer (128–132). (111). To modify the P. pastoris N-glycosylation pathway, the Some of the emerging mammalian cell line technologies are researchers performed the following steps. At first, they knocked discussed below. out the OCH1 (endogenous mannosyltransferase gene) using the gene disruption method (111). For efficient disruption, various Mammalian cells' engineering via siRNA vectors were designed; these vectors contained resistance genes Human IgG1 isotype contains two Asn-linked glycosylation for selection or selection was made on the basis of auxotrophy sites in its Fc region (133). Fc- mediated effector function is (112, 113). The OCH1 gene is involved in hyperglycosylation, as it influenced by the N-glycan attached to it (134, 135). As studies provides the outer branch for further glycosylation. After gene have shown, core fucose lacking glycan of the Fc region of disruption, UDP-Gal and UDP-GlcNAc transporters were intro- antibodies exhibits more efficiency than fucosylated antibodies, duced, which ensured the availability of sugar precursors in the both in vivo and in vitro (136–140). Unfortunately, almost all Golgi complex. At last, genes of mammalian origin involved in available therapeutic antibodies on the market are highly fuco- trimming and addition of sugar moieties such as glucanases and sylated, mostly containing fucose in their core oligosaccharide. In glycosyltransferases were introduced (111, 114, 115). mammalian cell lines, the fucosylation of glycoproteins is medi- Erythropoeitin (EPO) is a very important therapeutic glyco- ated by the a-1,6-fucosyltransferase (FUT8) gene, which transfers protein consisting of 165 amino acids. This protein has three N- fucose residue from GDP-fucose to GlcNAc in the N-glycan of glycosylation (Asn24, 38, 83) sites (116), which have a major role in glycoprotein (141). The substrate (GDP-fucose) of glycan fuco- its activity, secretion, and bioactivity in various types of cancer sylation is manufactured in the cytoplasm by both de novo and (117). Removal of N-glycan by mutagenesis resulted in a sub- salvage pathways. The de novo pathway, which contributes to most stantial decrease in bioactivity, stability, and hindrance in secre- of the intracellular GDP-fucose, has an enzyme, GDP-mannose tion. Similarly, N-glycans with no terminal sialic acids have their 4,6-dehydrate (GMD), involved in enzymatic reaction of the galactose exposed and are easily removed by galactose-specific pathway. The enzymes FUT8 and GMD can be important candi- receptors in serum (118). EPO, produced via GlycoFi technology, dates in controlling fucosylation of oligosaccharides (142, 143). has a human-like glycan and, when compared with EPO, has a In a study carried out by Imai-Nishiya and colleagues (111), yeast-like glycan (highly monosylated) in rat, showing remark- antibodies producing CHO cell lines to nonfucosylated antibo- able improvement in bioactivity and serum half-life (119). Hence, dies producing cells have been engineered without disturbing any the glycoproteins produced via GlycoFi technology were shown to characteristics of cells, except fucosylation. In their strategy, the have an advantage over those produced in wild-type yeast or less researchers used RNA interference with Lens culinaris agglutinin engineered yeast. (LCA) lectin as a phenotypic selection strategy. LCA lectin recognizes the a-1,6-fucosylated trimannose glycan core in cells and N-glycosylation Engineering Technologies commits them to apoptosis. For knockdown of the genes GMD and FUT8, constitutive vectors expressing siRNA against these for Mammalian Cells genes were introduced into an antibody producing CHO/DG44 Human cell lines allow human-like glycosylation of recombi- 32-05-12 cells (144, 145). Clones expressing a low level of nant anticancer proteins. This approach warrants that proteins targeted genes were selected for antibody analysis, which showed harbor at least nonimmunogenic glycans even then a lot of almost no fucosylation. They concluded that this strategy for promising technologies are being introduced to humanized controlling fucosylation of antibodies to enhance ADCC is quite recombinant therapeutics glycosylation (120). FDA has approved effective, economical, and less time consuming compared with many recombinant anticancer therapeutics produced in Chinese the use of homologous recombination for gene targeting in Hamster Ovary (CHO) cells (121, 122). Among these, pertuzu- mammalian somatic cells (145). This strategy has the potential mab (HER2 dimerization inhibitor), daratumumab (CD38-tar- for the development of next-generation antibodies for anticancer geted mAb), rituximab (anti-CD20 mAb), and siltuximab are just therapeutic use (146). some of the many examples used to treat breast cancer, relapsed multiple myeloma, non-Hodgkin B-cell lymphoma, and idio- GlycoExpress technology pathic multicentric Castleman disease, respectively (122–124). Glycotope GmbH, founded in 2001, developed novel technol- Among human cell lines, the HT-1080 (having fibrosarcoma ogies for production of biopharmaceuticals and then focused on origin) and the HEK293 (derived from human embryo kidney) an expression system that produces fully humanized glycopro- cells are used to manufacture glycosylated recombinant thera- teins. For this purpose, they developed GlycoExpress technology peutics. Agalsidase alfa, velaglucerase alfa rFVIIIFc, rFIXFc, epoetin (95), based on mammalian cell lines, which can produce and delta, and idursulfase are some of the many therapeutics pro- optimize humanized glycoproteins (147). Most of the mamma- duced in human cell lines. Additional recombinant therapeutics lian cell lines (e.g., CHO, BHK, or SP2/O) used for anticancer produced in the HuH-7 (hepatocellular carcinoma cells), HKB-11 therapeutics production can produce glycoproteins with glycans (Kidney/B Cell Hybrid), PER.C6 (Crucell), and CAP (CEVEC similar to those of humans but lacking a few important moieties, Amniocyte Production) human cell lines are currently being just as a-2,6-linked sialylationand bisecting GlcNAc are missing examined (125–127). Murine myeloma cell lines (NS0 and in these glycoproteins. On the contrary, few nonhuman addition- Sp2/0) have also been used for the production of recombinant al moieties are present, such as terminal NeuGc (a type of sialic anticancer mAbs such as elotuzumab (SLAMF7-directed immu- acid) or galactose attached to another galactose at the terminal nostimulatory antibody), cetuximab (inhibits EGFR), dinutuxi- position (148). These extra nonhuman components can increase mab (chimeric antibody), ofatumumab (anti-CD20 mAb), and the immunogenic response (149). To solve these problems,

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Glycotope used human cell lines in its GlycoExpress technology For stable modified cell line isolation, Concanavalin A (157) and further engineered them, as proteins are not glycosylated in was used for selection, which recognizes mannosylated, hybrid the same way in different types of cells. Different sets of cell lines and complex type N-glycans on cell surface proteins, leaving were formed to achieve different glycan profiles. behind GlycoDelete phenotypes. Later, it was found that 293S GlycoExpress cell lines are engineered using various techni- GlycoDelete cells are less adherent, which is favorable for sus- ques. The gene that needs to be removed is knocked out via gene- pension culturing (154). GM-CSF (158) and anti-CD20 were specific recombination, and then, the stable cell lines are isolated expressed in 293S GlycoDelete lines for N-glycan analysis. These after transfection with various glycosylation enzymes. Cells engi- cells were found to produce sialated trisaccarides or Gal-GlcNAc neered in the GlycoExpress toolbox can then address different disaccharides and rarely monosaccharide intermediates in con- steps of glycosylation. They can produce fucosylated or nonfu- trast to complex- and hybrid-type N-glycan by other types of cosylated, a-2,3 and a-2,6-sialylated or nonsialylated, highly mammalian cell lines. Meuris and colleagues also discovered that galactosylated, or nongalactosylated, and ranging from hybrid GlycoDelete anti-CD20 antibodies have a greater initial serum to complex-type glycoproteins. Glycoproteins with greater serum half-life than wild-type anti-CD20 in mice. This finding may be half-lives can be produced in these cells by sialylating them to very due to a decrease in sialated glycoproteins binding to lectin high degrees. Similarly, cell lines are available that can fucosylate receptors, which can lead to its clearance from the serum or sialylate glycoproteins in the range of 0% to naturally possible (159). Similarly, GlycoDelete antibodies showed more than maximum. However, this can be achieved by optimizing culturing 10-fold decrease in binding affinity to FcRs of humans, which is conditions and medium supplements (150–152). Antibodies are good, as safety is a concern in the case of neutralizing antibodies the most important class of biotherapeutics and the major target (159). In the case of antibodies binding to these receptors, the for glycol optimization. When IgG1 antibodies were produced in immune response is evoked and cytokine production is triggered. GlycoExpress cell lines, a 10- to250-fold increase was found in its Therefore, GlycoDelete technology favors production of anti- ADCC activity to improve anticancer therapy compared with bodies when the case is neutralization of antigen rather than those originally produced in rodent cell lines. Similar results were additional effector function. Likewise, the reduced N-glycanpro- found with other types of antibodies produced in GlycoExpress tocol is important in the biopharmaceutical industry, and the cell lines. In most cases, these results were obtained by fucose benefits of short N-glycans have also been reported (160–162). removal, addition of bisecting GlcNAc, and a high degree of galactosylation and sialylation. In one study, one type of antibody Conclusion and Future Prospects had enhanced ADCC activity without fucose removal (147). This study demonstrates that the perception that a-1,6-fucosylation Glycosylation is the most frequent posttranslational modifica- removal is the only way to improve ADCC to treat various types of tion of anticancer therapeutic proteins and therefore has a major cancers is incorrect. influence on biologic activity, specificity, and complexity, making them less immunogenic and well-tolerated. The safety profile and GlycoDelete technology high efficacy of these drugs has resulted in incredible growth in For efficient activity of glycoproteins, they need humanized and almost every area of medicine. Advancement of unique expression homogenous glycans (153). The heterogeneity of glycans in technologies, such as process optimization, modified hosts, pro- various expression systems is due to different steps of complex- moters, and secretion signals, has facilitated production of gram type N-glycan production. To achieve homogenous glycan pro- quantities of anticancer recombinant drugs at low cost and within duction, Meuris and colleagues introduced a technology called a short period of time. Among a variety of expression systems (e.g., GlycoDelete. This technology simplified and shortened the mam- yeast and mammalian cell lines) currently employed for produc- malian N-glycosylation pathway, leading to proteins with short tion of glycosylated anticancer therapeutics, mammalian-based and simple glycan carrying a sialylated trisaccharide. Cells pro- systems have been predominantly used. The major contributing duced as a result of GlycoDelete technology did not lose normal factors in selection of the expression system are the glycosylation physiologic processes or protein folding due to glycan modifica- composition and glycoforms or patterns. Anticancer glycopro- tion (154). Meuris and colleagues started GlycoDelete engineer- teins produced via GlycoFi technology in yeast show more resem- ing from human embryonic kidney 293S. These cells were defi- blance to their natural counterparts than those produced in less cient in carrying out N-acetylglucosaminyl transferase I (GnTI) engineered or wild-type yeast. Glycosylated anticancer therapeu- activity, which converts N-glycan into hybrid and complex type tics such as antibodies produced through GlycoExpress technol- glycan. GnTI-mutant cells [293SGnTI () cells] were previously ogy possess many folds increase in ADCC activity compared with produced by deleting GnTI (155). Then, these cells were trans- any other glycoengineering technology. Furthermore, antibodies fected with the fusion protein–containing fungus Hypocrea jecor- produced through GlycoDelete technology have a greater serum ina endoT (endo-b-N-acetylglucosaminidases; ref. 122) catalytic half-life and decreased binding affinity to humanFcRs, which domain and the human b-galactoside-a-2,6-sialyltransferase I enhances their safety profile. (ST6GALI; ref. 123) targeting domain for proper Golgi local- Despite the increasing number of glycosylation engineering ization. Endo T removes N-linked oligosaccharides and leaves technologies, along with their expression systems available for the glycoprotein with a single GlcNAc by breaking bonds use, there is no technology capable of meeting all challenges. between the first two GlcNAc residues (156). Then, this struc- Different glycosylation parameters (e.g., the glycan charge, ture is recognized by galactosyltransferases and sialyltrans- sequence, molecular size, and number of glycans attached) ferases, adding up galactose and sialic acid, respectively. can modulate the emerging technologies used in different Because of this GlycoDelete strategy, the N-glycosylation path- expression systems to different extents in the near future. The way remains confined to a three-step process, which success- significant potential of these technologies in different expres- fully homogenizes N-glycan (154). sion systems should lead to further research toward the

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development of anticancer therapeutic drugs with the lowest Disclosure of Potential Conflicts of Interest probability of contamination, high yield, inexpensive medium, No potential conflicts of interest were disclosed. human-like glycan isoforms, improving delivery of immune cells to tumor tissues, increasing antigen priming, and facili- Received January 18, 2018; revised March 22, 2018; accepted April 3, 2018; fi tating effector cell activation. published rst May 22, 2018.

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OF12 Cancer Res; 78(11) June 1, 2018 Cancer Research

Glycosylation of Recombinant Anticancer Therapeutics in Different Expression Systems with Emerging Technologies

Tariq Nadeem, Mohsin Ahmad Khan, Bushra Ijaz, et al.

Cancer Res Published OnlineFirst May 22, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-0032

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