Exploring human glycosylation for better therapies

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Molecular Aspects of Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m a m

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Exploring human glycosylation for better therapies

Q1 Larissa Krasnova a, Chi-Huey Wong a,b,* a

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Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA The Genomics Research Center, Academia Sinica, Taipei, Taiwan

A R T I C L E

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Article history: Received 1 March 2016 Revised 28 April 2016 Accepted 6 May 2016 Available online Keywords: Glycan antigens Glycan arrays Glycosylation probes Homogeneous antibodies Cancer vaccines

A B S T R A C T

Glycosylation of lipids and proteins is not encoded by genes directly and depends on many factors including the origin of cell-lines, differential expression of carbohydrate enzymes and availability of substrates, as well as environmental conditions. Individual cells from different tissues produce each glycoprotein as heterogeneous mixtures of glycoforms with distinct biological activities in response to different conditions and disease states. As the result, the study of glycosylation could not rely purely on biochemical methods; instead it requires a multidisciplinary approach utilizing a variety of methods including genetic manipulation and glycosylation pathway engineering, structural and functional proteomic analysis, chemical and enzymatic synthesis, development of glycosylation probes and glycan microarrays. This review highlights recent progress and demonstrates how the availability of structure-defined oligosaccharides enables development of new and improved therapies, such as therapeutic homogeneous antibodies and carbohydrate-based vaccines against cancer. © 2016 Published by Elsevier Ltd.

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Introduction .............................................................................................................................................................................................................................. 2 Glycan synthesis and automation ..................................................................................................................................................................................... 2 2.1. Programmable one-pot synthesis ........................................................................................................................................................................ 2 2.2. Automated solid-phase synthesis ........................................................................................................................................................................ 2 2.3. Enzymatic glycosylation coupled with co-factor regeneration ................................................................................................................. 3 2.4. Glycosidase mediated synthesis of oligosaccharides .................................................................................................................................... 4 Glycosylation probes .............................................................................................................................................................................................................. 5 Glycan microarrays ................................................................................................................................................................................................................. 6 Effect of glycosylation on the structure and function of protein: synthesis of homogeneous glycoproteins ......................................... 8 5.1. Mammalian N- and O-glycosylation ................................................................................................................................................................... 8 5.2. Effect of glycosylation on protein folding and stability ............................................................................................................................... 9 5.3. Synthesis of homogeneous glycoproteins ...................................................................................................................................................... 10 5.4. Synthesis of homogeneous glycoforms of monoclonal antibodies ........................................................................................................ 12 Development of carbohydrate-based vaccines for the treatment of cancer .................................................................................................... 13 6.1. Tumor-associated carbohydrate antigens (TACAs) ...................................................................................................................................... 14 6.2. Glycans on cancer stem cells .............................................................................................................................................................................. 14 6.3. Design of Globo H vaccine for the treatment of breast cancer: choice of carrier protein, adjuvant and design of unnatural (non-self) epitopes ............................................................................................................................................................................. 15

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* Corresponding author. Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA; The Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan. E-mail addresses: [email protected] or [email protected] (C.-H. Wong). http://dx.doi.org/10.1016/j.mam.2016.05.003 0098-2997/© 2016 Published by Elsevier Ltd.

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Conclusion and outlook ...................................................................................................................................................................................................... Disclosure statement ........................................................................................................................................................................................................... Acknowledgments ................................................................................................................................................................................................................ References ...............................................................................................................................................................................................................................

1. Introduction Oligosaccharides are often displayed on the cell surface through conjugation to lipids and proteins, and are involved in various intercellular communications and carbohydrate-mediated recognition processes. Since the biosynthesis of glycans depends on the expression of genomeencoded enzymes, the composition of cell-surface carbohydrates is specific to the cell type and alters as the cell undergoes developmental and functional changes. However, elucidation of the biological functions of glycans can be a complex task, and requires effective methods and tools for the study. Over the last 25 years, an impressive progress has been attained in our understanding the role of carbohydrates in biology (Committee, 2012). Much of this achievement is due to the improvements in the methods of oligosaccharides synthesis, which can deliver substantial amounts of pure material required for the functional study. Another element that expanded our knowledge of glycosylation events is the emergence of new tools for the study of glycosylation, such as methods for specific gene expression and sequencing, structural analysis and glycoproteomics, the design of glycosylation probes and glycan arrays. This made possible identification of novel glycan structures involved in various carbohydrate-mediated biological recognitions, which led to the development of carbohydrate-based medicines. 2. Glycan synthesis and automation One of the major challenges in oligosaccharide synthesis is selective glycosidic bond formation. The stereoselectivity of chemical glycosylation depends on the structural features of the glycosyl donors and protecting groups (PGs). Although many glycosylation procedures have been developed and provide a high degree of stereocontrol, their practicality remains a major challenge. Recent efforts have been directed toward the development of a simple and efficient method for glycan assembly, such as one-pot procedure for the construction of complex oligosaccharides (Raghavan and Kahne, 1993), including variations of this concept using the PG-controlled reactivity of glycosyl donors (Douglas et al., 1998; Huang et al., 2004), and the orthogonal one-pot strategy (Yamada et al., 1994) through highly specific activation of LGs (Yasomanee and Demchenko, 2013). 2.1. Programmable one-pot synthesis Although the one-pot strategy has proven to be quite efficient for the synthesis of complex oligosaccharides, it is limited to the laboratories with expertise in carbohydrate chemistry. To transform the highly specialized carbohydrate syntheses into a routine operation, a programmable strategy of oligosaccharide assembly has been developed

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by the Wong group (Zhang et al., 1999). This method utilizes designed software for the selection of thioglycoside building blocks donors based on their relative reactivity values (RRVs). The RRVs are determined from competition experiments against peracetylated thiomannoside (RRV=1) and reflect the effect of protecting groups on the reactivity of anomeric center. The current database contains more than 400 building blocks and has been successfully applied to the synthesis of various oligosaccharides, including colon cancer antigen Ley, sLex, fucosyl GM1, and embryonic stem cell surface carbohydrates Lc4 and IV2Fuc-Lc4 (Hsu et al., 2011). Fig. 1a illustrates synthesis of the Globo H antigen, which was performed in a highly efficient manner (Burkhart et al., 2001). 2.2. Automated solid-phase synthesis Development of automated oligosaccharide synthesizer has been highly anticipated for quite some time. The most advanced prototype developed by the Seeberger group utilizes reengineered peptide synthesizer with several adjustments to accommodate for glycosylation conditions, including temperature control of the reaction vessel and the use of inert gas atmosphere to handle sensitive reagents (Plante et al., 2001). As illustrated in the preparation of Globo H, the reducing end sugar is coupled to the solid support and is used as an initial glycosyl acceptor. The cycles of cou- Q4 pling – selective removal of temporary PG and generation of solid-bound acceptor – are repeated until the oligosaccharide of the desired length is assembled (Fig. 1b) (Werz et al., 2007). In general, each coupling step proceeds with high efficiency (80–90%) and can be monitored by UV trace of the Fmoc group. The release from solid support via Grubbs’ metathesis gives pentenyl glycoside donor. More recently, a new bifunctional linker was introduced, thus giving access to amine functionalized glycans suitable for immediate application, such as conjugation to the carrier protein for vaccine design or attachment to activated surfaces for glycan array fabrication (Krock et al., 2012). The main drawbacks of the method are the need for selective deprotection after each coupling step and the use of a large excess of building blocks. These drawbacks could be partially addressed by the solution phase automated synthesis using flow reactors (Geyer et al., 2009), fluorous tags (Jaipuri and Pohl, 2008; Zhang et al., 2009) or using reagent free donor activation as in the case of electrochemical synthesis of oligoglucosamines (Nokami et al., 2013, 2015). Nevertheless, the solid-phase automated synthesis was successfully applied to the synthesis of several notable targets including Ley, Lex antigens, short glycopeptides, glucosaminoglycan oligosaccharides, and GPI glycolipids (Seeberger, 2015). The idea of automated synthesis with enzymes immobilized on solid supports, coined artificial Golgi, was originally demonstrated by the Nishimura group (Nishimura, 2005), and inspired further studies toward enzymatic synthesis of

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Fig. 1. Different synthetic strategies towards preparation of Globo-H antigen: (a) programmable one-pot synthesis, (b) automated solid-phase synthesis, (c) enzymatic multi-gram synthesis of Globo H and SSEA-4 antigens with one-pot regeneration of sugar-nucleotides. Circle with clockwise arrows represent sugar-nucleotide regeneration system.

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oligosaccharides in the digital microfluidic devices (Martin et al., 2009; Ono et al., 2008). Additional improvements of automated methods include the use of polymers as solid supports, where solubility of solid phase is controlled by solvents or temperature (Huang et al., 2001). 2.3. Enzymatic glycosylation coupled with co-factor regeneration Although chemical methods are still commonly practiced in academic laboratories and are suitable for the

exploratory research, they are not always applicable to the large-scale synthesis. In nature oligosaccharides are assembled by enzymes; therefore, making enzymatic catalysis with glycosyltransferases (GTs) and glycosidases perfectly fit for the in vitro synthesis of complex oligosaccharides (Schmaltz et al., 2011). Many of mammalian GTs are of the Leloir type, which utilize nine basic nucleotide-activated building blocks (donors) for the stepwise synthesis of complex oligosaccharides. Microbial GTs are more suitable for in vitro synthesis as they can be easily expressed, scaled up and

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optimized to the desired substrate specificity (Chang et al., 2011; Lairson et al., 2008). The problems of using expensive nucleotide donors as substrates and feedback inhibition caused by the nucleoside phosphate by-products have been resolved with the development of nucleotidephosphate recycling system (Wong et al., 1982). Since then, multienzymatic protocols for the regeneration of other nucleotide donors have been developed (Cai, 2012) and applied to the synthesis of sialyl Lewis X (Ichikawa et al., 1992), disialyllacto-N-tetraose (Yu et al., 2014), heparin oligosaccharides (Zhou et al., 2011), hyauronic acid (De Luca et al., 1995) and other complex carbohydrates. The power of the efficient recycling systems has been demonstrated in the synthesis of Globo-H and SSEA-4 antigens on multigram scale (Fig. 1c) (Tsai et al., 2013b), which enabled translational research and clinical study of an experimen-

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tal vaccine for the treatment of breast cancer (completed Phase III). 2.4. Glycosidase mediated synthesis of oligosaccharides Glycosidases, or glycosyl hydrolases, are also suitable for the enzymatic synthesis of glycans under conditions, which are designed to shift equilibrium toward the product formation (e.g., use of activated glycosyl donors in excess, presence of organic co-solvent, etc). Depending on the site of glycoside cleavage, glycosidases can be of exo- or endotype. The synthetically relevant mechanisms of glycosidase catalysis are presented in Fig. 2a–d. Engineering of glycosyl synthases with abolished hydrolytic activity via mutation of the nucleophilic residue at the active site is one of the major breakthroughs in the

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Fig. 2. General mechanisms of enzymatic catalysis: (a) hydrolysis with retaining glycosidases and (b) synthesis with corresponding glycosyl synthases, (c) hydrolysis with substrate-assisted glycosidases and (d) synthesis with activated oxazoline donors. (e) In vitro glycan remodeling of expressed ribonuclease (RNase) with GTs (Witte et al., 1997) and endo-glycosidases (e.g., Endo M synthase mutant N175A) (Huang et al., 2009). (f) Commonly accepted abbreviations of monosaccharides and modifications used throughout this review: Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosesamine; GalNAc, N-acetylgalactosamine; Man, mannose; IduA, iduronic acid; Neu5Ac, N-acetylneuraminic acid; Fuc, fucose; S, sulfate.

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enzymatic synthesis of glycans (Fig. 2b) (Mackenzie et al., 1998). Selective site mutation and directed evolution techniques have also been used to generate synthetically valuable glycosyl synthases (Hancock et al., 2009). Currently, glycosidases and synthases of endo-type are the catalysts of choice for the preparation of homogeneous glycoproteins (Fig. 2e), such as therapeutic mAbs (Goodfellow et al., 2012; Huang et al., 2012; Lin et al., 2015; Zou et al., 2011).

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3. Glycosylation probes Cell-surface glycans are associated with numerous recognition processes in the biological system, and understanding the role of cell-surface glycans played in biology represents a significant challenge. One of the approaches to the study of glycosylation processes and identification of cell-surface glycans associated with cancer involve the design of carbohydrate-based probes, which can be divided into three categories: (a) glycosylation inhibitors that in-

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terfere with glycosylation pathways and lead to overexpression of a specific glycoform; (b) glycosylation probes that are taken as substrates and do not interfere with glycan biosynthesis, and can be used for glycan imaging and glycoprotein profiling; and (c) extracellular probes for cellsurface engineering (Fig. 3). Glycosylation probes that interfere with glycan biosynthesis and inhibitors of glycan processing enzymes represent an old-fashion approach toward the enrichment of a specific glycoform. Some of the classical examples include imminocyclitols, deoxymannojirimycin and kifunensin that act as α-mannosidase I inhibitors and can be used for the in vivo synthesis of high-mannose type glycoproteins (Esko and Bertozzi, 2009; Kiessling and Splain, 2010). More recent cases include the use of 3-Fax-Neu5Ac and 2-F-Fuc that allow remodeling of cell-surface glycans by inhibiting sialyl- and fucosyltransferases respectively (Fig. 3a) (Rillahan et al., 2012). In a separate study, 2-F-Fuc and 2-alkynyl-Fuc were successfully used for the production of fucose free

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Fig. 3. Graphic depiction of chemical probes designed for a specific application. (a) Inhibitors of GTs affect glycosylation profile of glycoconjugates (twisted line) displayed at the cell-surface. For example, 3-Fax-Neu5Ac inhibitor targets sialyltransferases and decreases sialic acid expression at cell-surface. Inhibitors of fucosyltransferases reduce expression of fucose. (b) Probes used for glycoprotein imaging and profiling. Sugar derivatives functionalized with alkyne or azide (sugars marked with white cross) are fed to the cell (step 1), where they are converted into sugar-nucleotides and introduced into proteins and lipids (step 2), which are then displayed at the cell-surface (step 3). After a certain incubation time, labeling of glycoconjugates (step 4) is then performed via bioorthogonal reaction between modified sugar and a reporter (green star with attached black cross), which can be a fluorescent dye or a biotinylated reagent. (c) Examples of extracellular probes for glycocalyx engineering.

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monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells (Okeley et al., 2013). In recent years, however, the gene-editing techniques were shown to provide good control over glycosylation pathways (Yang et al., 2015), therefore reducing the utility of chemical inhibitors for the production of glycoform-enriched proteins. The glycosylation probes that are designed as substrate analogs with a small reporter group for incorporation into glycoconjugates, and a following bioorthogonal reaction with complementary reagent, have been used in glycosylation imaging, profiling and cell-surface remodeling (Agard et al., 2004; Baskin et al., 2007; Friscourt et al., 2012; Ning et al., 2008; Rabuka et al., 2006; Sawa et al., 2006; Saxon and Bertozzi, 2000; Tanaka and Kohler, 2008). These probes were shown to be suitable for sialic acid pathways with N-acetylneuraminic acid and manosamine analogs as metabolic substrates, and for fucosylation with azido- or alkyne-Fuc probes, as well as glycosylation with GlcNAcprobe for the nucleo- and cytoplasmic O-GlcNAc modification of proteins (Fig. 3b) (Agard and Bertozzi, 2009; Du et al., 2009). In a typical experiment, treatment of cells with the mannosamine derivative bearing an azido group leads to incorporation of the azido-modified saialosides into the proteins and lipids, which are then displayed on the cell surface. Bioorthogonal reaction of the azide with another reagent containing an alkyne substituent allows labeling of sialosides with a detectable reporter. The acetyl protection of free hydroxyls of monosaccharide probe improves cell permeability of the probe and the protection is later cleaved by cytoplasmic esterases. The approach was greatly empowered by the introduction of the triazole forming click reaction, a fast, high-fidelity coupling reaction between biologically inert alkyne and azide groups. Following the original report on the fluorescent imaging of fucosylation in cancer cell lines (Hsu et al., 2007; Sawa et al., 2006), the click reaction-induced fluorescence became a general tool in glycoproteomics and glycan profiling. In glycoproteomics experiment, alkyne labeled glycoproteins are coupled with a biotin azide partner and subjected to a tryptic digest. At this point glycopeptides can be separated and hydrolyzed with peptide N-glycosidase (PNGase) to release the tagged glycans and Asp-peptides, thus permitting to identification of glycosylation sites by MS analysis. Next, the identified glycoprotein can be overexpressed in the disease-associated cell for analysis of the glycan structures. These data are then used for elucidation of the disease-associated glycan biomarkers and the glycosylated peptide sequences. Further validation study will require synthesis of identified glycoforms. Some of the examples of this technique include determination of the new N-glycosylation sites of the sialylated glycoproteome of prostate cancer cells (Hanson et al., 2007), N-glycosylation state of B-cell maturation antigen (Huang et al., 2013a), elucidation of the effect of EGFR sialylation on its dimerization and internal phosphorylation (Liu et al., 2011; Yen et al., 2015), and detection of Fuc-α(1–2)-Gal sequences (Chaubard et al., 2012). Additional sensing unit within a reporter molecule can lead to enhanced sensitivity of detection (Tsai et al., 2010), whereas the development of a bifunctional probe for dual imaging allows the study of intermolecular interactions on cell-surfaces (Feng et al., 2013). Moreover, inhibitors

equipped with a reporting tag can be used for the study of glycan processing enzymes. For example, the profiling of sialidases was performed using the alkyne modified 2,3difluoro-sialic acid (DFSA) (Tsai et al., 2013a). Bioorthogonal chemistries employed for the study of glycosylation include Staudinger ligation of triarylphosphines and azides (Saxon and Bertozzi, 2000), hydrazine and oxime formation reactions (Lemieux and Bertozzi, 1998; Luo et al., 2014) and several other useful transformations (McKay and Finn, 2014). Nonetheless, copper catalyzed click reaction outperforms other techniques in terms of efficiency, kinetics, selectivity and easy access to the coupling partners (Rostovtsev et al., 2002; Tornoe et al., 2002). The only drawback of Cu-click ligation is the cytotoxicity of copper ions, which prevents its use in live cells and organisms. This liability was addressed with the development of strainpromoted cycloaddition between cyclooctyne derivatives and azido-sugars (Chang and Bertozzi, 2012) and was successfully demonstrated during visualization of cellular O-glycosylation processes in zebra fish embryo (Laughlin et al., 2008). Despite great promise, these reagents suffer from low selectivity and slow kinetics, thus hampering the advancement of the real-time imaging. Reduction of background interference is another issue that is being addressed with the development of photo-induced electron transfer probes, such as new Azido-BODIPY (Shie et al., 2014) and dibenzocyclooctynol dyes (Friscourt et al., 2012). Cell-surface engineering is a new technology that employs synthetic glycoconjugate probes to study multivalent carbohydrate interactions in the context of cellular surfaces (Fig. 3c). Controlled modification of cell surface with carbohydrate epitopes can be used for the monitoring, study and regulation of specific biological responses. The examples of probes consist of polymers decorated with relatively short sialosides (Hudak et al., 2014; Paszek et al., 2014) and glycosaminoglycan-mimic polymers (Huang et al., 2014b; Pulsipher et al., 2014, 2015), which are covalently attached to the membrane-anchoring unit, either lipid or Hal-Tag protein. As expected, this new technique could be further empowered with the availability of a larger set of homogeneous glycan samples, which can become possible with the development of new efficient methods of glycan synthesis. 4. Glycan microarrays In an attempt to mimic glycan expression on cell surfaces, glycan array technology was introduced by three groups in 2002 (Fazio et al., 2002; Fukui et al., 2002; Wang et al., 2002) and has become a powerful tool for the highthroughput elucidation of glycan interactions with a variety of targets including glycan binding proteins (GBPs), antibodies, viruses and cells (Park et al., 2013; Rillahan and Paulson, 2011). Oligosaccharides immobilized on surfaces provide inexpensive study model of the multivalent representation of carbohydrate epitopes, which have low binding affinities in the monovalent form. Glycan array technology can be applied to a broad spectrum of research problems (Fig. 4). The progress in the area of glycan microarrays is associated with the development of new techniques for array fabrication, detection methods, assay design

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Fig. 4. Graphical depiction of glycan microarray and their use. (a) Example of non-covalent immobilization of glycans on Teflon-like coated ACG slides, which were applied for the profiling of glycosidases. Glycosidase treated slides can be directly subjected to MS-TOF analysis (Chang et al., 2010). (b) Binding activities of GBP (or glycosidases) is diminished in the presence of inhibitors, which can be used in a competition assay to screen libraries of compounds with inhibitory properties. (c) Glycan arrays allow evaluation of clustering effect and multi-ligand cross-reactivity. For example, mixed glycan arrays (binary mixtures) on AGC slides were used to study binding specificities of broadly neutralizing antibodies against HIV-1. Antibody PG9 showed strong preference for the mixture of bi-antennary complex-type and high-mannose type N-glycans, which confirmed presence of these two glycan structures at the binding epitope on gp120 of HIV-2 (Shivatare et al., 2016b). (d) Schematic depiction of glycan arrays composed of oligosaccharides isolated form natural sources and glycan structure interrogation by SGM approach. (e) In addition to GBP, cells, viruses and bacteria can be also used to study their affinity to specific oligosaccharide structures. (f) Application of glycan arrays for the diagnostics of cancer and microbial infections. For example, glycan arrays composed of tumor antigens can be used to test patient’s serum for the presence of specific antibodies, which may be an indication of the excising pathology.

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and most importantly glycan synthesis and library preparation (Geissner et al., 2014; Park et al., 2013; Rillahan and Paulson, 2011). Optimization of the methods of array fabrication, which covers choice of the solid support and immobilization procedures, is aimed at improving the stability of slides, cost efficiency, compatibility of slides with different detection methods and slide surface optimization to prevent false positive detection. Different chemistries and techniques for the glycan immobilization on solid supports have been developed (Park et al., 2013; Rillahan and Paulson, 2011). The most popular covalent attachment via amide linkage ensures the desired stability and control over the glycan presentation at the surface. An alternative and effective immobilization chemistry introduced recently by the Wong group involves attachment of phosphonic acid conjugated glycans onto the aluminum oxide-coated glass slide (ACG) (Chang et al., 2010). Arrays fabricated by this method permit a high control of glycan density and distribution, and produce an improved signal-to-noise ratio (Shivatare et al., 2016a). On the other hand, non-covalent immobilization of glycans allows its molecular mass identification (Fig. 4a) (Chang et al., 2010). Other combinations of the immobilization chemis-

try and solid supports, which are compatible with mass spectrometry, include the neolipid bilayers on indium-tin oxide (Beloqui et al., 2013) and self-assembled monolayers (SAMs) of PEG-thiols on gold (Ban et al., 2012). In the latter case, the SAMDI technique, which is a combination of SAMs and matrix-assisted laser desorption–ionization mass spectrometry, was applied to study activities of GTs (Fig. 4b). Procedures for quantification of the carbohydrate– protein interactions and determination of the surface dissociation constant (KD,surf), solution dissociation constant (KD) and Ki have been developed (Liang et al., 2007; Park et al., 2013). In a similar manner, the glycan microarrays can be applied for the high throughput screen of the new inhibitors of glycan processing enzymes that are involved in the biosynthesis of disease-associated epitopes. The array format also provides the means to assess the clustering effect of the carbohydrate epitopes (Godula and Bertozzi, 2012), density and the effects of multi-ligand cross-reactivity (Liang et al., 2011; Shivatare et al., 2016a). Compared to the traditional NHS array on glass slides, the ACG slide can be used to prepare more homogeneous arrays with controlled spatial design and mixed glycans for the study of multivalent hetero-ligand binding (Fig. 4c).

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The detection methods used for assessing glycan–GBP interactions include fluorescence microscopy, MALDI-TOF, surface plasmon resonance, quartz-crystal microbalance, radioactivity, oblique-incidence reflectivity difference microscopy, etc. The recently developed core-shell nanoparticle assay provides the enrichment, detection and signal amplification in the antibody screens with the sensitivities reaching the subattomole level (Liang et al., 2009). Depending on the research goal, the arrays can be composed of synthetic or isolated glycans. The latter are used for glycan structure elucidation. Of particular interest is a shotgun glycan microarray (SGM) approach, which is aimed at the parallel identification of both structure and function of physiologically relevant glycans (Fig. 4d) (Smith and Cummings, 2013). In the SGM method, natural glycans are isolated from a specific tissue or cell line, derivatized and purified. After preliminary characterization data is collected (MS, charge), a tagged glycan library is immobilized on the NHS-glass slides and screened for a desired function. In parallel, the same library is interrogated with a panel of lectins and antibodies of known specificities for terminal sugars and linkages. Digestion with exo-GH and the second round of interrogations with GBPs provide additional data, which is analyzed and compared against known structures. Currently, most of the natural glycan arrays are derived from N-glycans and glycolipids, as they can be selectively released by the action of PNGase F/A and ceramidases respectively. O-Glycan-derived natural arrays are less studied, due to the lack of general O-glycosidases and the nonselective nature of the chemical treatments, which gives cross-contamination of samples with N-glycan structures (Song et al., 2014). Structurally defined glycan arrays are used to profile specificities of a diverse range of GBPs including plant lectins, siglecs, galectins, antibodies, C-lectins, intact viruses, cells and bacteria (Fig. 4e). Perhaps the largest collection of structurally defined mammalian glycans is accumulated at the Consortium of Functional Glycomics (CFG) (Blixt et al., 2004). Established in 2001, the CFG library consists of >600 highly diverse structures that have been implemented in a variety Q5 of studies (http://www.functionalglycomics.org). Of particular interest to this review are microarrays composed of GSL oligosaccharides, which offer a powerful platform for monitoring the immune response to prostate cancer antigen RM2 (Chuang et al., 2013), Globo H (Huang et al., 2006) and SSEA-4 (Huang et al., 2013b). In addition, the microarrays composed of Globo H and SSEA-4 have been used for breast cancer diagnostics and vaccine validation (Huang et al., 2013b) (Fig. 4f). Another milestone in the field of welldefined glycan arrays is associated with the availability of diverse libraries of N-glycan oligosaccharides. Recently reported optimized chemoenzymatic routes toward N-glycan sequences has been already reported and with time should lead to full representation of all possible isomers (Li et al., 2015; Shivatare et al., 2016a; Wang et al., 2013b). 5. Effect of glycosylation on the structure and function of protein: synthesis of homogeneous glycoproteins Glycosylation of proteins is one of the most complex posttranslational modification events, which affects protein

folding, structure, function, stability and binding affinities. Protein glycosylation is classified into N- and O-glycosylations and are usually referred to static modification of proteins with complex oligosaccharide sequences (Fig. 5); such proteins are only found at the surface of the cell. However, O-glycosylation can technically include intracellular modification of proteins with β-GlcNAc (Torres and Hart, 1984), which plays a significant role in biological homeostasis and modulates cellular biology in response to variability of environmental conditions (Bond and Hanover, 2015; Lazarus et al., 2012, 2013; Ma and Hart, 2014). It is estimated that more than half of all human proteins are glycosylated by the competitive action of ~200 genome encoded GTs (not including other carbohydratemodifying enzymes) resulting in a formation of multiple glycoforms with distinct pharmacological profiles (Moremen et al., 2012). As a result, control of the glycosylation state of recombinant proteins becomes extremely important for understanding the effect of glycosylation on protein folding and function, and for developing glycoprotein pharmaceuticals (Datta et al., 2013; Sola and Griebenow, 2009). 5.1. Mammalian N- and O-glycosylation Protein glycosylation generally occurs co-translationally at the amide nitrogen of the Asn residue (N-glycosylation), or post-translationally at the hydroxy group of the Ser or Thr residues (O-glycosylation). N-Glycosylation commences in the endoplasmic reticulum (ER) via transferring of the oligosaccharide moiety from a lipid-linked substrate to the acceptor Asn-X-Ser/Thr (where X ≠ Pro) peptide sequons by oligosaccharyltransferase (OST) (Helenius and Aebi, 2001). After removal of the two terminal Glc residues, the GlcMan9GlcNAc2-linked polypeptide undergoes folding and a series of transformations associated within the protein quality control system (Helenius and Aebi, 2001; Moremen et al., 2012). The correctly folded proteins are then transferred for further processing to the Golgi apparatus, also a place of O-glycosylation. The subsequent trimming and modifications steps only leave the conserved Man3GlcNAc2-core, which is elongated with GlcNAc and Gal residues, and can be terminated by sialylation or fucosylation. The final compositions of N- and O-linked glycans are determined by the availability of sugar-nucleotide substrates and GTs. In addition to Asn- and Ser/Thrglycosylations, other less common protein modifications include C-mannosylation and O-mannosylation among others (Spiro, 2002). In broad terms, O-glycosylation can occur at the hydroxyl group of such amino acids as Ser, Thr, Tyr, hydroxy lysine, etc, and covers initial glycosylation with GalNAc, Gal, GlcNAc, Man, Fuc, Glc or Xyl (Steentoft et al., 2014). However, the mucin-type O-linked α-GalNAc oligosaccharides are the most common O-glycosylated proteins, which are expressed by mucosal epithelial cells (Linden et al., 2008; Perez-Vilar and Hill, 1999). In the healthy state, mucins fulfill an important function of a protective barrier to cells against mechanical damage and infections, act as decoys for bacterial adhesins and initiate intracellular signaling pathways in response to pathogen invasion (Linden et al., 2008). In addition, O-glycosylated proteins have been shown to play

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Fig. 5. Examples of the most abundant mammalian oligosaccharide sequences found in glycoproteins. (a) All N-linked glycans consist of the conserved core (Man3GlcNAc2). Depending on the composition of branches, N-glycans are divided into 3 types: high-mannose, hybrid and complex. Complex-type sequences are extended by GlcNAc or LacNAc repeat(s) and can be further modified by α-linked sialic acid, fucose, sulfation, etc. Possible modifications are shown by red lines and arrows. (b) O-linked glycans are classified into 8 types according to the conserved sequence. Cores 1–4 are the most common in healthy adults. Possible modifications are shown for cores 1–4 (red arrows).

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prominent roles in embryonic development, organogenesis and tissue homeostasis (Tabak, 2010; Tran and Ten Hagen, 2013). Compared to N-glycosylation, our understanding of O-glycosylation is limited. 5.2. Effect of glycosylation on protein folding and stability One of the most important roles of N-glycosylation is to assist proper folding of newly synthesized polypeptides during calnexin/calreticulin (CNX/CRT)-mediated folding cycle (Helenius and Aebi, 2001). As such, the intrinsic effect of glycosylation on stabilization of protein structure and folding kinetics has been assessed. Early reports showed the effects of glycans on the local peptide conformations (O’Conner and Imperiali, 1998). In contrast, N-glycosylation of folded protein does not seem to affect the average backbone fold; rather it provides a long-range stabilization of the tertiary or quaternary fold of glycoprotein (Imperiali and O’Connor, 1999; Wormald and Dwek, 1999). In a recent study, the Imperiali group investigated the effect of N-glycosylation (GlcNAc-GlcNAc) at different sites of bacterial immunity protein Im7, and observed most prominent stabilizing effect in compact turn motifs between segments of ordered structure, where glycosylation promotes folding and enhances the overall stability of the native protein (Chen et al., 2010). This study may explain why glycosylation is commonly identified at the transition

between different types of secondary structures and where it may be shield disordered sequences protecting them from proteolysis (Petrescu et al., 2004). Another general observation, which has emerged from the study of different glycoproteins, is the importance of first couple of glycan residues and their interactions with nearby amino acids (Wormald and Dwek, 1999). The effect was investigated using the cell adhesion and signaling molecule CD2, which is expressed on T-lymphocytes and natural killer cells. CD2 contains a 105 amino acid extracellular domain (hCD2ad), a single glycosylation site at Asn65 in the human homolog and has no disulfide bonds or proline residues to interfere with its folding. Systematic comparison of highmannose glycoforms suggests that the first GlcNAc residue attached to Asn is the most important component for structure stabilization. A total of ~3 kcal/mol of stabilization was attributed to the Man-GlcNAc-GlcNAc core trisaccharide. The first (form the reducing end) GlcNAc contributes 2/3 of the energy and the following Man-GlcNAc sugars provide 1/3 of the energy, whereas the outer sugars have insignificant effect on stabilization (Fig. 6). The results suggest that the core trisaccharide, which is highly conserved in the glycoproteins from eukaryotic species, is sufficient for acceleration of folding and stabilization in hCD2ad (Hanson et al., 2009). Further study of the system revealed that aromatic Phe residue at the [i-2] position to GlcNAc-Asn [i] provides an additional stabilization (Fig. 6) (Chen et al., 2013; Culyba

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Fig. 6. GlcNAc (1) speeds up folding by 0.8 kcal/mol. Stabilization of the folded structure by 2 kcal/mol is from GlcNAc (1) and an additional 1.1 kcal/ mol is from GlcNAc-Man (2–3) (Hanson et al., 2009). Phe at [i-2] provides additional stabilization of −0.7 to −2 kcal/mol (Culyba et al., 2011).

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et al., 2011). The apparent stabilizing interactions between the first GlcNAc residue and the aromatic substituent of nearby amino acid can explain an increased probability of aromatic residues in a position [i-1] and [i-2] (Petrescu et al., 2004). Moreover, introducing an aromatic residue at [i-2] position to Asn, so called enhanced aromatic sequons, increases glycosylation efficiency of OST and decreases N-glycan heterogeneity by suppressing further glycan processing in Golgi complex (Murray et al., 2015). In regard to monoclonal antibodies, the difference in thermodynamic stability of tertiary protein structure among different types of N-glycans has been studied for the Fc region of immunoglobulin (IgG1). Native Fc (complextype glycoform) was shown to have higher thermodynamic stability compared to its hybrid and high-mannose glycoforms, which were closer to endo-glycosidase treated Fc (Fc with mono GlcNAc) (Bowden et al., 2012). Dynamic O-glycosylation was also found to affect protein structure and stability. The O-GlcNAc-modification of RNA polymerase II was shown to initiate protein folding (Simanek et al., 1998). In a more recent report, O-GlcNAc-modification at Ser 75 of histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the polycomb repressive complex 2 was shown to play an essential role in its stability and enzymatic activity. EZH2 methyltransferase facilitates trimethylation of the K27 site of histone H3, resulting in the inhibition of tumor suppression (Chu et al., 2014). 5.3. Synthesis of homogeneous glycoproteins The synthetic task for the preparation of homogeneous glycoproteins usually consists of two parts: (a) synthesis of complex oligosaccharides and (b) site-specific conjugation of glycan to the polypeptide backbone. Currently, the established procedures for the chemical synthesis of N-linked oligosaccharide units still fail in terms of process scalabil-

ity and generality of the approach to allow straightforward access to a diverse set of structures (Walczak and Danishefsky, 2012; Walczak et al., 2013). However, chemical synthesis can be considerably simplified by the incorporation of enzymatic steps, e.g., late-stage introduction of challenging linkages, such as α2,3-/α2,6-sialylation of LacNAc and core fucosylation (Nycholat et al., 2013; Serna et al., 2011; Shivatare et al., 2013). Access to diverse sequences of N-glycans, particularly asymmetrically branched multiantennary structures, is essential for the development of new analytical and diagnostic tools. Making complex N-glycans available, especially the structures that are isomeric and non-distinguishable by mass spectrometry, is instrumental for the development of new methods of glycan sequencing. In addition, diverse welldefined N-glycan structures are needed for the preparation of comprehensive glycan arrays. To address the need for structure-defined N-glycans, Boons et al. reported a general Q6 strategy for the chemo-enzymatic synthesis of asymmetrically branched complex N-glycans, which takes advantage of the orthogonal protection of the branched trimannosyl core and selective modifications of PG-differentiated branches with GTs (Fig. 7a) (Wang et al., 2013b). In order to have access to the large sets of asymmetric glycans, a modular synthesis method, involving the chemo-enzymatic preparation of oligosaccharyl fluoride building blocks for the reaction with conserved ManGlcNAc2 core, has been developed (Fig. 7b) (Shivatare et al., 2016a). The only solution to the synthesis of homogeneous glycoproteins with well-defined glycan structures at multiple glycosylation sites is via ligation of glycopeptides (Payne and Wong, 2010; Unverzagt and Kajihara, 2013). In general, the ligation procedure requires an activated thioester intermediate, which can be obtained as intein-fusion protein through co-expression or as thioester by solid-phase peptide synthesis (SPPS), with glycan structure being installed by enzymatic or chemical methods. The next step involves activation of the thioester intermediate and coupling to the cysteine-terminated glycosylated peptide block via the native chemical ligation (NCL). Perhaps, the most significant example of NCL is the total synthesis of fully glycosylated homogeneous erythropoietin (EPO) realized by the Danishefsky group (Fig. 8a) (Wang et al., 2013a). The NCL method is advanced by the development of new orthogonal protecting groups for thiols, masked thioesters or unnatural thiolated amino acids, which after the ligation step can be converted to the native structures (Payne and Wong, 2010). This approach allows more flexibility during the design of the synthetic strategy, as cysteine is not very common and can be missing at the desired ligation junctions. In many cases, desulfurization reaction is performed (e.g., Cys → Ala); however, Cys → Ser transformation has also been reported (Okamoto et al., 2009). In this regard sugarassisted ligation (SAL) provides a convenient way of assembling glycopeptide fragments (Brik et al., 2006, Yang et al., 2007 #155) (Fig. 8b). Modification of the acetamido Q7 moiety of GalNAc (or GlcNAc) with a sulfhydryl group enables transthioesterification with the peptide thioester, which triggers S→N acyl transfer resulting in a peptide bond formation. The thio-auxiliary or the entire monosaccharide unit can then be selectively removed. A major advantage

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Fig. 7. (a) Synthesis of the asymmetrically branched N-glycans with GTs starting form orthogonally protected oligosaccharide (Wang et al., 2013b). (b) Retrosynthetic disconnections that illustrate modular synthesis of N-glycans. (c) In modular approach oligosaccharide branches are synthesized chemoenzymatically, protected and sequentially coupled to the ManGlcNAc2 core (Shivatare et al., 2016b).

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of the SAL method is its broad sequence tolerance at ligation sites (Ficht et al., 2007; Payne et al., 2007). Other useful techniques include the thiol auxiliary-based cysteine-free ligation and protease-assisted ligations (Payne and Wong, 2010; Schmaltz et al., 2011). Unfortunately, chemical synthesis is not suitable for the large-scale production of glycoproteins of commercial value, and as expected, the manufacturing of glycoprotein pharmaceuticals is currently carried out in mammalian systems (Hossler et al., 2009), mainly Chinese hamster ovary (CHO) cell lines, which employ human-like glycosylation pathways (Sola and Griebenow, 2009). The manipulation of biosynthetic pathways of glycoproteins in mammalian cells is performed by a combination of standard techniques, including genetic mutation, knockout and pathway inhibition (Schmaltz et al., 2011). The most commonly used process has been the production of afucosylated recombinant glycoproteins via deletion of fut8 or inhibition of the GDPfucose synthesis pathway, and controlled sialylation of glycan structures predominantly with human Neu5Ac. However, with the emergence of new precision genome editing methods, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), as well as the RNA-guided clustered regularly interspaced short palin-

dromic repeat (CRISP)/Cas nuclease system, it becomes much easier to dissect glycosylation pathways and biological functions of complex oligosaccharides by a genetic approach (Steentoft et al., 2014). ZFN technique has been already used for generation of cell lines with simplified glycosylations, which helped characterization of O-GalNAc (Steentoft et al., 2011; Yang et al., 2014) and O-Man glycoproteoms (Vester-Christensen et al., 2013). More recently, the same grouped performed systematic ZFN knockout screen of glycosyltransferase genes in CHO cells, using human EPO as recombinant reporter, and identified key glycogens that control decisive steps during N-glycosylation, therefore providing basis for construction of desirable, more homogeneous glycosylation capacities (Yang et al., 2015). Glycoengineering in yeast (De Pourcq et al., 2010) and bacterial cultures (Merritt et al., 2013) can also provide improved cost-efficiency and prevent potential contamination with mammalian borne pathogens (Fig. 9c). To date, the most successful adaptation of humanized pathways was achieved for N-glycosylation with complex bi-antennary structures in Pichia pastoris strains (Vogl et al., 2013). Unfortunately, engineering of the humanized O-glycosylation pathways in yeast is problematic. While glycoengineering of cell lines and host strains may provide a high degree of glycan

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Fig. 8. (a) Chemical synthesis of homogeneous EPO by NCL (Wang et al., 2013a) and (b) SAL step in the synthesis of diptericin (Yang et al., 2007).

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homogeneity, the effort is mainly regulatory to ensure safety and efficacy. 5.4. Synthesis of homogeneous glycoforms of monoclonal antibodies The identification and optimization of therapeutic glycoforms require synthesis of glycoproteins in a pure, homogeneous form. For example, the degree of sialylation of human erythropoietin (hEPO) was found to correlate with serum stability, which led to the development of novel erythropoiesis stimulating protein, Aranesp®. The stability of EPO was improved by increasing the number of sialic acids (from ~14 to ~22) via introduction of two additional N-glycosylation sites (Egrie and Browne, 2001). However, asialoEPO was shown to have potent neuroprotective activity (Erbayraktar et al., 2003). The therapeutic monoclonal antibody Rituximab, produced in CHO cells, contains more than 50 glycan structures at Asn297 of the Fc region, and different glycoforms exhibit different functions (Shade and Anthony, 2013; Vidarsson et al., 2014). For example, removal of the core-fucose enhances its binding to the FcγIIIa receptor (FcγRIIIa) (Ferrara et al., 2011) and leads to the increase of effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) (Fig. 9a) and complement-dependent cytotoxicity (CDC) (Anthony et al., 2008; Kaneko et al., 2006). On the other hand, the anti-inflammatory activity of IgG requires α2,6-sialylation of the glycan on IgG, which depends on glycan interactions with FcγRIIb receptor (Lin et al., 2015).

In vitro remodeling of oligosaccharide sequences of recombinant proteins with glycosidase is well suited for the preparation of glycoproteins with high degree of glycan homogeneity (Fig. 2a) and can be readily accomplished in two steps by trimming glycans of a protein to a single GlcNAc residue with endoglycosidases (e.g., Endo H, S or F) and subsequent modification of GlcNAc with GTs (Witte et al., 1997) or glycan reconstruction via transglycosylation reaction (Takegawa et al., 1995). Synthesis with endo-glycosidases (ENGases) and mutated synthases coupled with the use of activated oxazoline donors has become particularly effective for the preparation of homogeneous glycoproteins and glycopeptides (Huang et al., 2012; Lin et al., 2015). Enabled by this approach, the synthesis of several notable IgG1 targets including a universal glycan for optimized effector functions was performed (Fig. 9b) (Lin et al., 2015). The last example illustrates the specificity of Endo S, which permits manipulations of the complex fucosylated glycans on IgG substrates (Goodfellow et al., 2012). Several other ENGases have been reported over the years of which the most synthetically useful include high mannose-specific Endo-H, Endo A and Endo M (Schmaltz et al., 2011). The latter can cleave most N-glycan structures, but prefers the complex type for transglycosylation reaction. Most recently, the Wang group reported Endo F3 D165A mutant, which has unique specificity for tri-antennary complex-type N-glycans and can transfer oxazoline donor onto GlcNAc(α1,6Fuc) of short peptide, as well as GlcNAc(α1,6Fuc) of rituximab (Giddens et al., 2016). Despite the generality of the approach, ENGase-mediated synthesis requires excess of oxazoline donor in order to

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Fig. 9. (a) Illustration of ADCC mechanism. (b) Enzymatic in vitro glycan remodeling of Rituximab with endo-glycosidases and synthesis of optimized glycoform with α2,6-sialylated complex-type N-glycan (Lin et al., 2015).

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achieve high conversions, making it expensive and leading to non-specific modification of other nucleophilic residues of proteins with oxazoline substrate (Parsons et al., 2016). Therefore, alternative strategies should be developed to overcome these drawbacks. 6. Development of carbohydrate-based vaccines for the treatment of cancer The ability of the immune system to discriminate foreign antigens from self-expressed epitopes has been exploited for the development of carbohydrate-based vaccines (Anish et al., 2014; Astronomo and Burton, 2010; Berti and Adamo, 2013). While early vaccines utilize capsular polysaccharides isolated from pathogens, current efforts are focused on the development of structurally defined synthetic vaccines in order to improve efficacy and safety. Quimi-Hib® is the first example of a synthetic vaccine that entered the Cuban vaccination program in 2004 (Fig. 10a). Conjugation of the carbohydrate antigen to human serum albumin (HSA) or more immunogenic tetanus toxoid (TT) resulted in the induction of long-term protective antibody titers against Haemophilis influenzae type B (Hib) with a 99.7% success rate in children. Another promising direction has been the development of GPI-based antiparasitic vaccines (Anish et al., 2014; Tsai et al., 2012), particularly against malaria parasite Plasmodium falciparum (Fig. 10b) (Schofield et al., 2002). Though the search for an

HIV vaccine based on 2G12 interactions with gp120 met little success, the recent discovery of glycan-dependent broadly neutralizing antibodies (bNAbs) opens an opportunity to the rational design and development of a new class of vaccines. Understanding the structural interactions between bNAbs (PG9 and PG16) and viral gp120 provides insights into the design and synthesis of the V1V2 glycopeptide epitopes and helps identify the necessary structural features for the efficient binding of PG9 and PG16 antibodies (Amin et al., 2013; Aussedat et al., 2013; Wang, 2013). In addition to classical preventive vaccines against human pathogens, there has been an emergence of therapeutic vaccines targeting cancer cells with altered glycosylation states. However, identification of the carbohydrate epitopes exclusively and specifically expressed on the surface of cancer cells represents a major challenge. In addition, most glycans are poor immunogens that only stimulate a B-cell dependent immune response and generate IgM antibodies with short memory. Conjugation of a carbohydrate antigen to a protein carrier provides a means of engaging T-cell help and activating the adaptive immune system to induce a longlasting immune response. Also, with a proper design of adjuvant, the generated cytokines, such as IL-4, could interact with B cells to induce a class switch from IgM to IgG isotypes. Overall, the strategy for vaccine design encompasses the identification of non-self carbohydrate epitopes, optimization of conjugation chemistry and linker between

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Fig. 10. (a) Structure of Quimi-Hib® vaccine (Verez-Bencomo et al., 2004) and (b) vaccine candidate based on GPI of Plasmodium falciparum against malaria infection (Schofield et al., 2002).

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glycan epitope and carrier protein, selection of optimal carrier protein and adjuvant for vaccine administration. However, despite continuing progress, the development of carbohydrate-based cancer vaccines has not been successful so far, perhaps due to the difficulty in the identification and validation of non-self epitopes, and a poor understanding of vaccine uptake, processing and presentation, as well as antibody class switch. 6.1. Tumor-associated carbohydrate antigens (TACAs) The development of anticancer vaccines requires the identification of unique epitope markers, which are specific to cancer cells. The molecules on the surface of cancer cells are often self-antigens and just overexpressed, therefore they cannot be used as epitopes for vaccine design, because the antibodies generated from such vaccines may also attack normal cells and cause autoimmune response. This is particularly true if proteins are used as epitopes. However, as cell-surface carbohydrates are assembled by many enzymes, during the progression of cancer some unique glycan structures may be differentially assembled and expressed on the surface of cancer cells. Hakomori et al. have pioneered the discovery of glycolipids, which are overexpressed on the surface of various cancer cells. This type of glycans has been termed as tumor-associated car-

bohydrate antigens (TACAs) (Hakomori and Young, 1978). Since then, the list of TACAs identified and explored in vaccine design includes blood-group antigens (sLex, Lex, LeyLex, sLea, Lea, Ley), gangliosides (Globo H, GM2 GD2, GD3, fucosyl GM1) and mucin O-glycans (Tn, TF, sTn) (Astronomo and Burton, 2010). Unfortunately, previous attempts to develop cancer vaccines based on TACAs have failed, although development of therapeutic antibodies targeting TACAs has met some success. Recent studies in our group have shown that the three globo-series glycolipids, namely SSEA3, SSEA4 and Globo H (Fig. 11a), are such unique markers on the cell surface of breast cancer cells and the cancer stem cells, as well as on the cell surface of other 15 different types of cancers, including lung cancer, pancreatic cancer, colon cancer, etc (Chang et al., 2008; Lou et al., 2014). This finding has raised the hope to develop therapeutic vaccines against cancers. 6.2. Glycans on cancer stem cells The discovery of cancer stem cells (CSCs), a cell subpopulation with self-renewing and tumor-growing abilities, and plasticity in heterogeneous cancer tissues (Bomken et al., 2010; Clarke et al., 2006), has stimulated interests in understanding cancer progression and developing new therapies and methods of early diagnosis (Beck and Blanpain,

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Fig. 11. (a) Structures of SSEA-3, SSEA-4 and Globo H. (b) Structure of C34 adjuvant. (c) Second generation anti-cancer vaccine based on Globo H antigen and DT-197 carrier. (d) Globo H vaccines bearing non-native fucose with azide modification (Lee et al., 2014).

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2013; Jordan et al., 2006; Pece et al., 2010). The markers currently used for the enrichment of cancer stem cells, however, are not specific enough, resulting in an incomplete understanding of cancer stem cells. Recent studies have shown that the breast cancer stem cells (BCSCs) isolated with CD44+CD24-/lowSSEA-3+ or ESAhiPROCRhiSSEA-3+ formed more mammospheres and resulted in better tumor formation, than the cells with ESAhiPROCRhi or CD44+CD24-/low (Cheung et al., 2016). As few as 10 cells with CD44+CD24-/low SSEA-3+ were able to form tumor in mice model, compared to 100 cells with CD44+CD24-/low. It appears that SSEA-3 is a BCSC marker that plays a major role in cancer progression. Moreover, it was observed that by manipulating the expression of β-1,3galactosyltransferase 5(β3GalT5) in cancer cells, one can control the cell surface levels of SSEA-3, SSEA-4 and Globo H, as well as the cellular survival and tumorigenicity. Interestingly, knockdown of β3GalT5 in cancer cells could trigger both apoptosis and inhibition of cell proliferation through different mechanisms, as a caspase-3 null cell line MCF-7 underwent a limited level of apoptosis and profound suppression of cell growth after knockdown of β3GalT5. On the contrary, in normal mammary epithelial cells, which lack SSEA-3 on the cell surface, knockdown of β3GalT5 did not affect these phenotypes. This study reveals that SSEA-3 is a novel glycan marker for enrichment of BCSCs, and both SSEA3 and β3GalT5 are also potential new targets for the development of breast cancer therapeutics. These findings are further supported by the study of antibodies designed to target the globo series glycans (Chang et al., 2008; Hakomori, 1985; Hakomori and Young, 1978; Huang et al., 2013b; Kannagi et al., 1983; Lou et al., 2014), and by the late-stage clinical trials of a cancer vaccine for the treatment of metastatic breast cancer (Danishefsky et al., 2015).

6.3. Design of Globo H vaccine for the treatment of breast cancer: choice of carrier protein, adjuvant and design of unnatural (non-self) epitopes Due to the low immunogenic properties of carbohydrate antigens, the choice of the protein carrier and adjuvant becomes essential. The most commonly used carriers include diphtheria toxoid (DT), tetanus toxoid (TT), human serum albumin (HSA) and keyhole limpet hemocyanin (KLH). Various linkers and conjugation chemistries can be used for the covalent attachment of glycan epitope to the carrier provided they do not stimulate non-specific antibody production. Switching from the protein carriers to synthetic peptides or lipid antigens is another step to reduce the variability of vaccine composition caused by the incomplete conjugation reactions (Cavallari et al., 2014). Another challenge in the development of carbohydratebased vaccines is that glycans are T-cell independent and often produce IgM type of antibodies with short-term memory and weak effector functions. A class switch from IgM to IgG in B cells is thus necessary to make the vaccine more effective with long-term memory, and this goal has been achieved with the use of a designed glycolipid adjuvant to stimulate the immune response and to induce a class switch (Huang et al., 2013b). The development of a carbohydrate-based vaccine for metastatic breast cancer, which included the selection and synthesis of Globo H as the target, the development of the vaccine conjugate and adjuvant design for class switch, and the analysis of Globo H distribution on the surface of various cancer cells, cancer stem cells and normal cells have been reported (Danishefsky et al., 2015). The first synthesis of Globo H was accomplished through the use of glycal chemistry, and the synthetic Globo H was conjugated to the carrier

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protein KLH and combined with QS21 adjuvant for evaluation of the vaccine in phase I human trials for the treatment of metastatic breast cancer (Wilson and Danishefsky, 2013). The programmable one-pot synthesis method was then introduced, which enabled the mid-stage proof-of-concept phase II trial and an initial phase III trial (Burkhart et al., 2001). Finally, enzymatic synthesis of Globo-H coupled with cofactor regeneration was used for the late-stage multicenter trials and manufacture of the product (Tsai et al., 2013b). Along this path of development, it was discovered that a new α-galactosylceramide-type (C34) adjuvant (Fig. 11b) designed to target the CD1d receptor on dendritic cells and B cells was able to activate natural killer (NK) T cells (NKT cells) to induce both Th1 and Th2 responses including a class switch from IgM to IgG (Huang et al., 2014a). A new generation of Globo H vaccine was thus developed using DT as carrier and C34 as adjuvant (Fig. 11c), which was shown to induce IgG antibodies to target the three globo series glycans, indicating its broad use as anticancer therapy (Huang et al., 2013b). A historical perspective on development of anti-cancer Globo H vaccine from chemical bench to clinical studies can be found in a recent review (Danishefsky et al., 2015). In an attempt to improve the efficacy of the current formulation and to study the effect of non-self glycan antigens on stimulation of immune system, a series of modified Globo H-DT conjugates were synthesized and evaluated for their ability to elicit IgG. Among them, DT conjugated to the Globo H derivative with artificial azido group modification at the C-6 position of the non-reducing end fucose demonstrated a robust IgG immune response, much greater than that of the parent unmodified Globo H-DT conjugate (Fig. 10d) (Lee et al., 2014). 7. Conclusion and outlook The development of practical methods for the synthesis of complex oligosaccharides facilitated identification of novel and unique carbohydrate structures on the surface of cancer cells. This interesting class of molecules enabled investigation of their functions associated with the cancer initiation and progression, and has led to the development of early diagnostic methods, and therapeutics, including antibodies and vaccines for the treatment and prevention of cancer. Disclosure statement The authors are not aware of any affiliation, biases, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. Acknowledgments We thank the financial support provided by Academia

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Chi-Huey Wong Professor Wong is currently the President of Academia Sinica, Taiwan, and Professor of Chemistry at the Scripps Research Institute. He received his BS and MS from National Taiwan University, and Ph.D. in chemistry from MIT in 1982. After one year of post-doctoral training at Harvard University, he taught at Texas A&M University for 6 years, then became the Ernest Hahn Chair in Chemistry at the Scripps Research Institute until 2006, when he was also President of Academia Sinica. His research interests are in the field of chemical biology with focus on the development of new methods for glycoscience research. He is a recipient of the American Chemical Society Arthur C. Cope Medal, The Royal Society of Chemistry Robert Robinson Award, and the Wolf Prize in Chemistry, and is a member of the US National Academy of Sciences.

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Larissa Krasnova is a Staff Scientist in the laboratory of Prof. Chi-Huey Wong at The Scripps Research Institute, La Jolla. She received her BS from Moscow State University and her Ph.D. in chemistry from University of Toronto. Her current research interests involve synthesis of small molecules as antiinfective agents and probes for the study of glycosylation processes.

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