Glycan Epitopes Research Articles

Immunization with Outer Membrane Vesicles Displaying Designer Glycotopes Yields Class-Switched, Glycan-Specific Antibodies

The development of antibodies against specific glycan epitopes poses a significant challenge due to difficulties obtaining desired glycans at sufficient quantity and purity, and the fact that glycans are usually weakly immunogenic. To address this challenge, we leveraged the potent immunostimulatoryactivity of bacterial outer membrane vesicles (OMVs) to deliver designer glycan epitopes to the immune system. This approach involved heterologous expression of two clinically important glycans, namely polysialic acid (PSA) and Thomsen-Friedenreich antigen (T antigen) in hypervesiculating strains of non-pathogenic Escherichia coli. The resulting glycOMVs displayed structural mimics of PSA or T antigen on their surfaces, and induced high titers of glycan-specific IgG antibodies following immunization in mice. In the case of PSA glycOMVs, serum antibodies potently killed Neisseria meningitidis serogroup B (MenB), whose outer capsule is PSA, ina serum bactericidal assay. These findings demonstrate the potential of glycOMVs for inducing class-switched, humoral immune responses against glycan antigens.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

Carbohydrate specific T cell stimulation by HIV envelope glycoprotein

Abstract HIV has been a major threat to human health and a protective AIDS vaccine has not as yet been developed. The design of the current generation of vaccines does not make use of specific scientific knowledge to maximize stimulation of critical immune cells (i.e., helper T cells) involved in producing protective antibodies. HIV-1 surface is decorated with a heavily glycosylated envelope protein called gp120, whose interaction with the CD4 molecule is the key step for the virus’s entry into CD4+ T cells. Gp120 glycans have been shown to play critical roles in Env structure, viral infectivity, antigenicity, receptor binding and HIV-1 transmission. Our goal is to identify CD4+ T cell repertoires recognizing the glycan shield of HIV-1 envelope glycoprotein, gp120. We have gained significant evidence for glycan/glycopeptide presentation on the antigen presenting cell (APC) surfaces serving as potential epitopes to induce carbohydrate-specific T cells (Tcarb) response. Additionally, we demonstrate that certain populations of CD4+ T cells differentiate between glycosylated and non-glycosylated gp120 epitopes, suggesting carbohydrate recognition by CD4+ T cells. Using the discovery of such Tcarbs and their glycan epitopes, we can design and develop knowledge-based, new-generation HIV vaccines that will elicit strong and long lasting immune response to protect from HIV. We believe our study will yield a new platform to develop a new-generation of protective HIV vaccines.

Translational glycobiology: Patient-oriented glycoscience research.

In his 1945 report to President Harry S. Truman entitled “Science: The Endless Frontier”, Vannever Bush, the then-Director of the USOffice of Scientific Research and Development, outlined his vision for a “National Research Foundation”, which ultimately became the “National Science Foundation”. In that document, Bush wanted to distinguish “basic research” from “applied research”, and offered a plan to create a government-sponsored funding agency to promote scientific education and to provide grants to nonprofit organizations for the pursuit of basic research: “Basic research is performedwithout thought of practical ends. It results in general knowledge and an understanding of nature and its laws. This general knowledge provides the means of answering a large number of important practical problems, though it may not give a complete specific answer to any one of them. The function of applied research is to provide such complete answers” (Bush 1945). In drawing the distinction between basic and applied research, Bush emphasized that the latter is problem-inspired. Thus, applied research expressly seeks to provide solutions to specific problems, i.e., is goal-oriented to offer “complete answers” to “practical ends”. Applied research that is devoted specifically to addressing unmet medical needs is called “translational research”. Analogous to other “-omics” (e.g., proteomics and genomics), glycomics is the comprehensive investigation of the structures, interactive pathways, and dynamic changes of glycans that impact biological systems, including both physiologic and pathologic processes. However, in the strictest sense, the term “glycomics” refers principally to the categorization/ characterization/elucidation of the structures of glycomolecules (e.g., encompassing information generated via NMR and mass spectrometry analysis), while the expression “glycosciences” focuses on how glycan structures relate to biologic and chemical processes/properties. Seamlessly bridging these two realms, there exists a discipline which I call “translational glycobiology”, a term, which I hold, refers to glycoscience-based research that is specifically motivated by the intent to alleviate human suffering, which is distinct from types of glycomics inquiry that are not patient-oriented and, also, differs from the application of an existing glycoscience-based technique or reagent in clinical medicine simply because someone clever realized its utility in that setting. In this thematic special issue of Glycobiology, the field of translational glycobiology is highlighted through the first-person narratives of four authors, each of whom is an experienced clinician. It is hoped that these articles will be both enlightening and entertaining, as each author was encouraged to relay their personal research journey within the pertinent historical perspective(s). These authors represent four distinct areas of clinical medicine: (i) Cell therapeutics (Sackstein); (ii) Transplant surgery (Cooper); (iii) Internal medicine/Allergy & Immunology (Bochner); and (iv) Dermatology (Maytin). The first article provides a framework to evaluate the biologic activity of glycoconjugates, reviews the discovery of the CD44 glycoform known as “HCELL”, and discusses how cell surface glycans can be custommodified to optimize cellular delivery to predetermined anatomic sites and thereby enable the application of cell-based therapeutics. The second article addresses how glycans impact solid organ transplantation, and how strategies to alter expression of critical glycan determinants may pave the way to cross-species transplantation (i.e., xenotransplantation), thus overcoming the critical shortage of organs needed to save the lives of patients with organ failure. The third article describes the discovery of a sialoadhesin now known as “Siglec-8” and how our increasing understanding of the structure and biology of this molecule is yielding novel therapies for allergy and other immunologic diseases. The fourth article reviews our current information on the complex biology of the glycosaminoglycan hyaluronic acid, and how structural modifications of this molecule can be harnessed to improve wound healing and dampen inflammation, effects far broader than its more recognized role in cosmetic applications as a spacefiller/wrinkle-remover. There is no prerequisite that investigators in translational glycobiology possess MD degrees. Indeed, at present, most of the efforts in translational glycobiology are being undertaken by scientists with Ph.D. degrees, essentially all of which have had prescribed preparation for careers in glycoscience. However, the contributing authors for this theme issue were chosen specifically because they are each accomplished clinicians, and, notably, because each did not have formal training in the discipline of glycoscience. Inspired to find solutions for alleviating the suffering of their patients, each clinician Glycobiology, 2016, vol. 26, no. 6, 544–545 doi: 10.1093/glycob/cww035 Introduction

Bacteriophage Tailspikes and Bacterial O-Antigens as a Model System to Study Weak-Affinity Protein-Polysaccharide Interactions.

Understanding interactions of bacterial surface polysaccharides with receptor protein scaffolds is important for the development of antibiotic therapies. The corresponding protein recognition domains frequently form low-affinity complexes with polysaccharides that are difficult to address with experimental techniques due to the conformational flexibility of the polysaccharide. In this work, we studied the tailspike protein (TSP) of the bacteriophage Sf6. Sf6TSP binds and hydrolyzes the high-rhamnose, serotype Y O-antigen polysaccharide of the Gram-negative bacterium Shigella flexneri (S.flexneri) as a first step of bacteriophage infection. Spectroscopic analyses and enzymatic cleavage assays confirmed that Sf6TSP binds long stretches of this polysaccharide. Crystal structure analysis and saturation transfer difference (STD) NMR spectroscopy using an enhanced method to interpret the data permitted the detailed description of affinity contributions and flexibility in an Sf6TSP-octasaccharide complex. Dodecasaccharide fragments corresponding to three repeating units of the O-antigen in complex with Sf6TSP were studied computationally by molecular dynamics simulations. They showed that distortion away from the low-energy solution conformation found in the octasaccharide complex is necessary for ligand binding. This is in agreement with a weak-affinity functional polysaccharide-protein contact that facilitates correct placement and thus hydrolysis of the polysaccharide close to the catalytic residues. Our simulations stress that the flexibility of glycan epitopes together with a small number of specific protein contacts provide the driving force for Sf6TSP-polysaccharide complex formation in an overall weak-affinity interaction system.

Broadly neutralizing antibody specificities detected in the genital tract of HIV-1 infected women.

Broadly neutralizing antibodies (bNAbs) targeting conserved epitopes on the HIV envelope glycoprotein have been identified in blood from HIV-1 infected women. We investigated whether antibodies in the genital tract from these women share similar epitope specificities and functional profiles as those in blood. Immunoglobulin (Ig)G and IgA antibodies were isolated from cervicovaginal lavages or Softcups from 13 HIV-infected women in the CAPRISA cohort using Protein G and Peptide M, respectively. Binding antibodies to envelope antigens were quantified by ELISA and binding antibody multiplex assay. Neutralizing antibody titers and epitope targets were measured using the TZM-bl assay with Env-pseudotyped wild-type and mutated viruses. HIV-specific IgG, but not IgA, was detected in genital secretions and the ratio of total IgG to HIV-specific IgG was similar to plasma. HIV-specific IgG reacted with multiple envelope antigens, including V1V2, gp120, gp140 and gp41. Two women had high plasma titers of HIV-specific IgG3 which was also detected in their genital tract samples. IgG from the genital tract had neutralizing activity against both Tier 1 and Tier 2 primary HIV-isolates. Antibodies targeting well known glycan epitopes and the membrane proximal region of gp41 were detected in genital secretions, and matched specificities in plasma. Women with plasma bNAbs have overlapping specificities in their genital secretions, indicating that these predominantly IgG isotype antibodies may transudate from blood to the genital tract. These data provide evidence that induction of systemic HIV-specific bNAbs can lead to antiviral immunity at the portal of entry.

ChemInform Abstract: One‐Pot Multienzyme (OPME) Systems for Chemoenzymatic Synthesis of Carbohydrates

AbstractReview: 89 refs.

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis.

At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody.

Two non-human glycan epitopes, galactose-α-1,3-galactose (α-gal) and Neu5Gc-α-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while α-gal attached to Fc glycans was not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Neu5Gc antibody resided in a small subset of mAbs carrying two or more Fc Neu5Gc, while mAbs harboring only one Neu5Gc showed no reactivity. Since most Neu5Gc epitopes were distributed singly on the Fc of mAbs, our results suggest that the potential antigenicity of Fc Neu5Gc is low. Our study could be referenced in the process design and optimization of mAb production in murine myeloma cells and in the quality control of mAbs for industries and regulatory authorities.

Pre-existing Antibody: Biotherapeutic Modality-Based Review.

Pre-existing antibodies to biotherapeutic drugs have been detected in drug-naïve subjects for a variety of biotherapeutic modalities. Pre-existing antibodies are immunoglobulins that are either specific or cross-reacting with a protein or glycan epitopes on a biotherapeutic compound. Although the exact cause for pre-existing antibodies is often unknown, environmental exposures to non-human proteins, glycans, and structurally similar products are frequently proposed as factors. Clinical consequences of the pre-existing antibodies vary from an adverse effect on patient safety to no impact at all and remain highly dependent on the biotherapeutic drug modality and therapeutic indication. As such, pre-existing antibodies are viewed as an immunogenicity risk factor requiring a careful evaluation. Herein, the relationships between biotherapeutic modalities to the nature, prevalence, and clinical consequences of pre-existing antibodies are reviewed. Initial evidence for pre-existing antibody is often identified during anti-drug antibody (ADA) assay development. Other interfering factors known to cause false ADA positive signal, including circulating multimeric drug target, rheumatoid factors, and heterophilic antibodies, are discussed.

Identification of the binding roles of terminal and internal glycan epitopes using enzymatically synthesized N-glycans containing tandem epitopes.

Glycans play diverse roles in a wide range of biological processes. Research on glycan-binding events is essential for learning their biological and pathological functions. However, the functions of terminal and internal glycan epitopes exhibited during binding with glycan-binding proteins (GBPs) and/or viruses need to be further identified. Therefore, a focused library of 36 biantennary asparagine (Asn)-linked glycans with some presenting tandem glycan epitopes was synthesized via a combined Core Isolation/Enzymatic Extension (CIEE) and one-pot multienzyme (OPME) synthetic strategy. These N-glycans include those containing a terminal sialyl N-acetyllactosamine (LacNAc), sialyl Lewis x (sLex) and Siaα2-8-Siaα2-3/6-R structures with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) sialic acid form, LacNAc, Lewis x (Lex), α-Gal, and Galα1-3-Lex; and tandem epitopes including α-Gal, Lex, Galα1-3-Lex, LacNAc, and sialyl LacNAc, presented with an internal sialyl LacNAc or 1-2 repeats of an internal LacNAc or Lex component. They were synthesized in milligram-scale, purified to over 98% purity, and used to prepare a glycan microarray. Binding studies using selected plant lectins, antibodies, and viruses demonstrated, for the first time, that when interpreting the binding between glycans and GBPs/viruses, not only the structure of the terminal glycan epitopes, but also the internal epitopes and/or modifications of terminal epitopes needs to be taken into account.

Correlative Fluorescence and Scanning Electron Microscopy of Labelled Core Fucosylated Glycans Using Cryosections Mounted on Carbon-Patterned Glass Slides.

The aim of the study is co-localization of N-glycans with fucose attached to N-acetylglucosamine in α1,3 linkage, that belong to immunogenic carbohydrate epitopes in humans, and N-glycans with α1,6-core fucose typical for mammalian type of N-linked glycosylation. Both glycan epitopes were labelled in cryosections of salivary glands isolated from the tick Ixodes ricinus. Salivary glands secrete during feeding many bioactive molecules and influence both successful feeding and transmission of tick-borne pathogens. For accurate and reliable localization of labelled glycans in both fluorescence and scanning electron microscopes, we used carbon imprints of finder or indexed EM grids on glass slides. We discuss if the topographical images can provide information about labelled structures, the working setting of the field-emission scanning electron microscope and the influence of the detector selection (a below-the-lens Autrata improved YAG detector of back-scattered electrons; in-lens and conventional Everhart-Thornley detectors of secondary electrons) on the imaging of gold nanoparticles, quantum dots and osmium-stained membranes.

Arabinogalactan proteins have deep roots in eukaryotes: identification of genes and epitopes in brown algae and their role in Fucus serratus embryo development.

Arabinogalactan proteins (AGPs) are highly glycosylated, hydroxyproline-rich proteins found at the cell surface of plants, where they play key roles in developmental processes. Brown algae are marine, multicellular, photosynthetic eukaryotes. They belong to the phylum Stramenopiles, which is unrelated to land plants and green algae (Chloroplastida). Brown algae share common evolutionary features with other multicellular organisms, including a carbohydrate-rich cell wall. They differ markedly from plants in their cell wall composition, and AGPs have not been reported in brown algae. Here we investigated the presence of chimeric AGP-like core proteins in this lineage. We report that the genome sequence of the brown algal model Ectocarpus siliculosus encodes AGP protein backbone motifs, in a gene context that differs considerably from what is known in land plants. We showed the occurrence of AGP glycan epitopes in a range of brown algal cell wall extracts. We demonstrated that these chimeric AGP-like core proteins are developmentally regulated in embryos of the order Fucales and showed that AGP loss of function seriously impairs the course of early embryogenesis. Our findings shine a new light on the role of AGPs in cell wall sensing and raise questions about the origin and evolution of AGPs in eukaryotes.

Immunological Approaches to Biomass Characterization and Utilization.

Plant biomass is the major renewable feedstock resource for sustainable generation of alternative transportation fuels to replace fossil carbon-derived fuels. Lignocellulosic cell walls are the principal component of plant biomass. Hence, a detailed understanding of plant cell wall structure and biosynthesis is an important aspect of bioenergy research. Cell walls are dynamic in their composition and structure, varying considerably among different organs, cells, and developmental stages of plants. Hence, tools are needed that are highly efficient and broadly applicable at various levels of plant biomass-based bioenergy research. The use of plant cell wall glycan-directed probes has seen increasing use over the past decade as an excellent approach for the detailed characterization of cell walls. Large collections of such probes directed against most major cell wall glycans are currently available worldwide. The largest and most diverse set of such probes consists of cell wall glycan-directed monoclonal antibodies (McAbs). These McAbs can be used as immunological probes to comprehensively monitor the overall presence, extractability, and distribution patterns among cell types of most major cell wall glycan epitopes using two mutually complementary immunological approaches, glycome profiling (an in vitro platform) and immunolocalization (an in situ platform). Significant progress has been made recently in the overall understanding of plant biomass structure, composition, and modifications with the application of these immunological approaches. This review focuses on such advances made in plant biomass analyses across diverse areas of bioenergy research.

Immunolocalization of cell wall carbohydrate epitopes in seaweeds: presence of land plant epitopes in Fucus vesiculosus L. (Phaeophyceae).

Land plant cell wall glycan epitopes are present in Fucus vesiculosus. RG-I/AG mAbs recognize distinct glycan epitopes in structurally different galactans, and 3-linked glucans are also present in the cell walls. Cell wall-directed monoclonal antibodies (mAbs) have given increased knowledge of fundamental land plant processes but are not extensively used to study seaweeds. We profiled the brown seaweed Fucus vesiculosus glycome employing 155 mAbs that recognize predominantly vascular plant cell wall glycan components. The resulting profile was used to inform in situ labeling studies. Several of the mAbs recognized and bound to epitopes present in different thallus parts of Fucus vesiculosus. Antibodies recognizing arabinogalactan epitopes were divided into four groups based on their immunolocalization patterns. Group 1 bound to the stipe, blade, and receptacles. Group 2 bound to the antheridia, oogonia and paraphyses. Group 3 recognized antheridia cell walls and Group 4 localized on the antheridia inner wall and oogonia mesochite. This study reveals that epitopes present in vascular plant cell walls are also present in brown seaweeds. Furthermore, the diverse in situ localization patterns of the RG-I/AG clade mAbs suggest that these mAbs likely detect distinct epitopes present in structurally different galactans. In addition, 3-linked glucans were also detected throughout the cell walls of the algal tissues, using the β-glucan-directed LAMP mAb. Our results give insights into cell wall evolution, and diversify the available tools for the study of brown seaweed cell walls.

Measuring Multivalent Binding Interactions by Isothermal Titration Calorimetry.

Multivalent glycoconjugate-protein interactions are central to many important biological processes. Isothermal titration calorimetry (ITC) can potentially reveal the molecular and thermodynamic basis of such interactions. However, calorimetric investigation of multivalency is challenging. Binding of multivalent glycoconjugates to proteins (lectins) often leads to a stoichiometry-dependent precipitation process due to noncovalent cross-linking between the reactants. Precipitation during ITC titration severely affects the quality of the baseline as well as the signals. Hence, the resulting thermodynamic data are not dependable. We have made some modifications to address this problem and successfully studied multivalent glycoconjugate binding to lectins. We have also modified the Hill plot equation to analyze high quality ITC raw data obtained from multivalent binding. As described in this chapter, ITC-driven thermodynamic parameters and Hill plot analysis of ITC raw data can provide valuable information about the molecular mechanism of multivalent lectin-glycoconjugate interactions. The methods described herein revealed (i) the importance of functional valence of multivalent glycoconjugates, (ii) that favorable entropic effects contribute to the enhanced affinities associated with multivalent binding, (iii) that with the progression of lectin binding, the microscopic affinities of the glycan epitopes of a multivalent glycoconjugate decrease (negative cooperativity), (iv) that lectin binding to multivalent glycoconjugates, especially to mucins, involves internal diffusion jumps, (bind and jump) and (v) that scaffolds of glycoconjugates influence their entropy of binding.

Protein-Carbohydrate Interactions, and Beyond …

Carbohydrates are ubiquitous and play an intriguing role inside the cell as well as on the cell surface.[...]

Databases of Conformations and NMR Structures of Glycan Determinants.

The present study reports a comprehensive nuclear magnetic resonance (NMR) characterization and a systematic conformational sampling of the conformational preferences of 170 glycan moieties of glycosphingolipids as produced in large-scale quantities by bacterial fermentation. These glycans span across a variety of families including the blood group antigens (A, B and O), core structures (Types 1, 2 and 4), fucosylated oligosaccharides (core and lacto-series), sialylated oligosaccharides (Types 1 and 2), Lewis antigens, GPI-anchors and globosides. A complementary set of about 100 glycan determinants occurring in glycoproteins and glycosaminoglycans has also been structurally characterized using molecular mechanics-based computation. The experimental and computational data generated are organized in two relational databases that can be queried by the user through a user-friendly search engine. The NMR ((1)H and (13)C, COSY, TOCSY, HMQC, HMBC correlation) spectra and 3D structures are available for visualization and download in commonly used structure formats. Emphasis has been given to the use of a common nomenclature for the structural encoding of the carbohydrates and each glycan molecule is described by four different types of representations in order to cope with the different usages in chemistry and biology. These web-based databases were developed with non-proprietary software and are open access for the scientific community available at http://glyco3d.cermav.cnrs.fr.

Abstract 4983: GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer

Abstract Purpose: MUC4 is one of the membrane-bound mucins that is expressed de novo during pancreatic cancer (PC) and contributes to pathogenesis. Further, MUC4 is aberrantly glycosylated in PC and some of its tumor-promoting effects are, in part, mediated by interactions facilitated by the glycan epitopes, which are generally masked under normal conditions. Mucins predominantly undergo O-glycosylation that is initiated by a family of enzymes known as GALNTs (UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase), catalyzing the transfer of first sugar residue (GalNAc) to ser/thr residues in mucin tandem repeat domains. Not much is known about the specific glycosyltransferases (GTs) involved in MUC4 glycosylation. Understanding the specific enzymes regulating differential glycosylation of MUC4 in cancer may be important for developing therapeutic strategies for modulating MUC4 function in PC. Thus, the goal of this study was to identify specific GALNT(s) catalyzing MUC4 glycosylation and investigate its role in differential MUC4 glycosylation in PC. Materials & Methods: PCR array analysis was performed in a panel of MUC4 expressing and non-expressing PC cell lines, normal pancreatic cancer cells and MUC4 expressing normal colon cells to identify differentially expressed GTs. To determine the altered glycosylation of MUC4 in normal versus cancerous conditions, Co-immunoprecipitation (Co-IP) studies were conducted using several carbohydrate-specific antibodies. Stable knockdown of GALNT3 was performed in PC and normal colon cells to understand its involvement in differential MUC4 glycosylation. To determine the functional implications of GALNT3 mediated aberrant glycosylation, various in vitro assays such as colony formation and migration were carried out. Results: PCR array and immunoblot analysis demonstrated GALNT3 as one of the GTs upregulated in MUC4 expressing cells as compared to MUC4 non-expressing PC cells. Interestingly, Co-IP analysis revealed differential expression of carbohydrates epitopes such as STn, SLex, SLea on MUC4 in PC and normal colon cells. GALNT3 knockdown in cancer cells resulted in decreased expression of Tn but simultaneous increase in STn epitope. Interestingly, we observed significant increase in colony formation and migration in GALNT3 knockdown cells. This was associated with increase in proliferation and EMT markers in GALNT3 knockdown PC cells such as N-cadherin, vimentin and phosphorylated ErbBs. Conclusion: Overall, our results suggested a correlation of MUC4 and GALNT3 expression, and knockdown of GALNT3 resulted in altered MUC4 glycosylation rather than complete abrogation, suggesting a possible compensatory increase in other GALNTs and other GTs. Further investigations along these lines will provide insight into specific GTs regulating MUC4 glycosylation and glycan epitope-mediated MUC4 functions in cancer. Citation Format: Seema Chugh, Vinayaga Srinivasan Gnanapragassam, Maneesh Jain, Moorthy Ponnusamy, Surinder Kumar Batra. GALNT3 mediated differential MUC4 glycosylation in the progression of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4983. doi:10.1158/1538-7445.AM2015-4983

Abstract 2485: Novel monoclonal antibody targeting cancer stem cells

Abstract Caerus Therapeutics (Caerus) is developing a monoclonal antibody directed to a cancer specific epitope on CD66c (CEACAM6) designated 109.12. CD66c has been shown in preclinical studies to be expressed on the colorectal cancer stem cell population but not on normal intestinal stem cells, making this a potential target for a therapeutic antibody. Previous attempts by other companies to develop anti-CD66c antibodies were unsuccessful due to cross-reactivity with CD66c on normal tissues. Caerus has developed a monoclonal antibody to a cancer-specific, glycan epitope that is only expressed in the adult during malignant transformation, enabling the targeting of CD66c on both cancer stem cells (CSCs) as well as bulk tumor without cross-reactivity with CD66c on normal cells. The glycan epitope recognized by the antibody has a novel structure not previously described that is highly protein-specific and site-specific. The specificity is due to a unique dual-recognition determinant for the site-specific glycosylation. In addition to its expression on colorectal tumorspheres, the epitope recognized by the 109.12 antibody is expressed by a wide variety of cancers including colon, pancreatic, lung stomach and a subtype of breast cancer. The biological role of CD66c in malignancy and tumor dissemination is well documented; the specificity of the antibody for a CD66c epitope only expressed in malignant cells offers multiple opportunities for immunotherapy and diagnosis of a diverse spectrum of cancers. Citation Format: Jeff Holderness, Amy Turner, Sam dolezal, Steve Wolpe, Michael Pierce, Cohava Gelber. Novel monoclonal antibody targeting cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2485. doi:10.1158/1538-7445.AM2015-2485