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EMBARGOED FOR RELEASE: Tuesday, Aug. 21, 2012, 2:15 p.m. Eastern TimeNote to journalists: Please report that this research was presented at a meeting of the American Chemical Society.
Newswise — PHILADELPHIA, Aug. 21, 2012 — As a step toward designing the first effective vaccine against HIV, the virus that causes AIDS, scientists are reporting new insights into how a family of rare, highly potent antibodies bind to HIV and neutralize it — stop it from infecting human cells. The antibodies were isolated from people infected with HIV and work against a wide range of HIV strains. The researchers described the study today at the 244th National Meeting & Exposition of the American Chemical Society, the world’s largest scientific society.
The report was part of a symposium titled “Glycoscience at the Crossroad of Health, Materials and Energy.” It focused on one of the newest and hottest areas of research, named for glycans chains of sugars that coat the outer surface of cells in the body like icing on a cake. Glycans are key to various activities, helping cells communicate with one another, for instance. The slightest miscue in glycan formation can result in serious diseases. Reports in the symposium focused on advances toward using knowledge about glycans in medicine and other fields.
“The difficulty in preparing a vaccine against HIV lies in the fact that it is ‘variable,’ meaning there are many strains and subtypes, all of which all slightly different,” explained Ian Wilson, D.Sc., who presented the study. “Recently, researchers have identified antibodies that can recognize many subtypes of HIV, not just one. The antibodies came from a few HIV-infected patients out of thousands. If we can determine what parts of the virus those antibodies are binding, then we could use that information to design a single vaccine that could protect people against most or all of the HIV strains and subtypes.”
Worldwide, about 34 million people are infected and living with HIV. About 2.7 million people are newly infected every year — these are infections that an anti-HIV vaccine could prevent. Efforts to develop a vaccine have thwarted scientists and consumed the money of funding agencies and drug companies for almost 30 years.
Vaccines typically consist of a piece of a virus or a killed or inactive version of a virus, which is injected. This “antigen” causes the person’s immune system to make antibodies against the virus. But two types of HIV exist — HIV-1 (the most common form) and HIV-2. Four groups of HIV-1 are known, and at least nine subtypes of Group M (which causes about 90 percent of all HIV-1 infections) exist. And all of these types and groups and subtypes are different; a vaccine designed against one of them would not necessarily work against other subtypes, complicating vaccine development.
Usually, antibodies target proteins of a virus. But HIV is a wolf in sheep’s clothing — it hides its precious proteins under a sugary shield, which is formed from the infected person’s own sugars. That way, the immune system often doesn’t even realize a foreign virus is lurking in its midst.
As a step toward making an effective and broadly neutralizing vaccine, Wilson and colleagues at the IAVI Neutralizing Antibody Center at the Scripps Research Institute analyzed antibodies that have the surprising ability to bind to many subtypes of HIV-1. These antibodies, called PG and PGTs, were isolated in previous work from HIV-infected individuals. His team figured out which parts of HIV several of these antibodies were binding. These unusually potent antibodies can bind to and neutralize up to 80 percent of HIV-1 virus types. Therefore, a vaccine that could prompt the body to make these antibodies would offer wide-ranging protection for an immunized person.
“The new PG and PGT antibodies are relatively rare antibodies that can actually bind to sugars on the glycan shield,” explained Wilson. “But they go even farther. They get through the shield of sugars and manage to contact the protein below.”
It turns out that many of these special antibodies bind to two glycans and a particular portion of a protein to which those sugars attach. “Once we understand this binding, then we can try to design an immunogen, or antigen, that would elicit the production of those antibodies in humans,” he said. Therefore, a vaccine may someday contain two glycans and a piece of protein that resembles the binding site for the PG and PGT antibodies. The researchers in the field call this process “structure-assisted vaccine design.”
Wilson said that it is unclear when a vaccine based on this work would become available. However, the finding that sugars are instrumental to this process just goes to underscore the importance of studying sugars in the laboratory.
The researchers acknowledged funding from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R01AI084817 and P01AI082362, as well as from the International AIDS Vaccine Initiative (IAVI).
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Abstracts from the symposium “Glycoscience at the Crossroad of Health, Materials, and Energy” follow.
Abstracts
Broadly neutralizing antibodies that recognize the HIV-1 Env glycan shieldIan A. Wilson1, Dr., D.Phil., 10550 N Torrey Pines Road, BCC206, La Jolla, CA, 92037, United States, 858-784-9706, [email protected]
The isolation of highly potent antibodies that recognize a broad diversity of HIV-1 clades has opened up tremendous opportunities for enhancing our understanding of how to neutralize HIV-1. Crystal structures of antibodies have been determined that recognize different epitopes including the gp41 membrane proximal region, the gp120 receptor binding site and clusters of high mannose glycans on gp120. New human monoclonal antibodies (PG and PGT families) have been discovered recently that potently neutralize 70-90% of HIV-1 isolates across all clades. These exciting new antibodies were derived from direct functional screening of B cells from IAVI protocol G donors (Theraclone/Monogram) and are unusually potent. These antibodies have unique features and bind to novel epitopes that include the glycan shield as well as segments of gp120. Structural characterization of these broadly neutralizing antibodies, as well as identification of their glycan epitopes on HIV-1 Env, provide tremendous insights for neutralization of HIV-1 and can now be used for structure-assisted vaccine design.
This work was carried out in collaboration with the Burton lab at TSRI and IAW was supported by NIH GM46192, the International AIDS Vaccine Initiative (IAVI) and NIH AI84817.
Interplay of carbohydrate chemistry and translational medicine Chi-Huey Wong1,2, Academia Sinica, 128 Academia Road, Section 2, Nankang District, Taipei, N/A, 115, Taiwan Republic of China, +886-2-2789-9400, +886-2-2785-3852, [email protected]
Protein glycosylation is one of the most complex post-translational events. According to the prediction based on the Asn-X-Ser/Thr sequon, more than 90% of human proteins are glycosylated. However, the real number is unknown. The significance of glycosylation at the molecular level is not well understood, and as such the pace for the development of carbohydrate-based drug discovery and diagnosis is relatively slow. It is thus important to develop new tools to study the effect of glycosylation on the structure and function of proteins and other biologically active molecules. This lecture will focus on the development of new methods for the synthesis of oligosaccharides and homogeneous glycoproteins with well defined glycan structure, study of glycosylation effect on protein folding, development of glycan arrays for the high-throughput analysis of protein-glycan interaction and design of glycoprotein vaccines to tackle the problems of influenza infection and cancer progression.
Challenges and opportunities for plant glycosciences in energy Markus Pauly1, Prof., Calvin Lab, Berkeley, CA, United States, 510-642-1722, [email protected]
All cells of higher plants are encased in a mainly carbohydrate based cell wall also referred to as lignocellulosics. Lignocellulosics represent the dominant carbon sequestration system on land as their production amount in just 2 days equals the entire annual production of all chemicals combined made by humans. However, lignocellulosics are not merely a carbon storage sink for plants, but they represent sophisticated, highly complex materials that plants produce to benefit their sessile life-styles allowing their survival for up to several millennia as is the case in some tree species. Lignocellulosics, the by far dominant component of plant biomass, consists of a complex aggregate of microcrystalline cellulose microfibrils, crosslinking water-soluble hemicelluloses, and a water repellent polyphenol, lignin.
In order to utilize this renewable resource for biofuels or other commodity chemicals it is desirable to understand how the plant synthesizes these polymers. Such knowledge may also one day lead to tailoring the polymer structures for numerous applications in varying industries.
Various strategies have been employed in our lab to identify the genes involved in the synthesis of this polymer including massive parallel pyrosequencing of relevant plants and tissues, forward genetic screens for plants with altered wall polymer structures, and reverse genetic approaches of analyzing insertional knock-out lines. The results demonstrate that plants could be identified/generated that harbor advantageous attributes for biorefineries. The ultimate goal of this research is to one day understand the complete biosynthetic pathway of a wall polymer.
Improving vaccines with natural antibodiesSteven J Sucheck1, Professor, PhD, The University of Toledo, Department of Chemistry, MS602, 2801 West Bancroft Street, Toledo, OH, 43606, United States, 419-530-1504, 419-530-1990, [email protected]
It has been shown that antigens conjugated to the cognate ligands of natural antibodies (NAs) show improved immunogenicity. Therefore, we designed a vaccine that contained a tumor-related glycopeptide fragment from the variable number tandem repeat (VNTR) region of MUC1. The target glycopeptide was prepared by solid phase peptide synthesis and contained an N-terminal azido moiety for click conjugation with a synthetic alkynyl derivative of the Toll-like receptor ligand and vaccine adjuvant Pam3Cys. The NA ligand, L-rhamnose, was conjugated with cholesterol using a tetraethylene glycol (TEG) linker. The two components were associated by formulation into a liposome by the extrusion method using the glycosylated Pam3Cys-MUC1 conjugate, L-rhamnose-TEG-cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine ( DPPC) in a total lipid concentration of 30 mM. The formulated liposomes demonstrated binding with both anti-rhamnose and mouse anti-human MUC1 antibodies. Results of an ongoing immunological evaluation of the vaccine will also be discussed.
Chemoenzymatic synthesis and analysis of glycosaminoglycansRobert J Linhardt1, Professor, PhD, 110 Eighth Street, Troy, NY, United States, 518-276-3404, [email protected]
Glycosaminoglycans (GAGs) are anionic polysaccharides of critical importance in human biology. Heparin, the most highly sulfated GAG has been used as an anticoagulant for over 75 years. Currently heparin and related low molecular weight heparins are prepared by extraction from porcine tissue. We have developed an efficient chemoenzymatic synthesis of heparin from a non-animal source. The varying sulfo group patterns make heparin chain an extremely challenging target. Here, we show our efforts to synthesize heparin, low molecular weight heparin and ultra-low molecular weight heparins using a chemoenzymatic approach relying on biosynthetic enzymes, including heparosan synthases, sulfotransferases and epimerase. Chemoenzymatic synthesis is reliable over a wide range of scales and should be useful in both research and pharmaceutical applications. The disaccharide composition and sequence of the products obtained are then characterized by using electrospray ionization mass and mass/mass spectrometry.
Nanoscale field-effect transistor biosensors for label-free detection of carbohydrate-protein interactions Guo-Jun Zhang1,2,, Prof., PhD, Hubei University of Chinese Medicine, School of Laboratory Medicine, 1 Huangjia Lake West Road, Wuhan, Hubei, 430065, China, 86-27-68890070, 86-27-68890071, [email protected]
In this presentation, we developed, for the first time, the silicon nanowire (SiNW)-based biosensor capable of label-free electrically detecting carbohydrate-protein interactions with high sensitivity by covalently immobilizing un-modified carbohydrates on the sensor surface. To avoid modifying carbohydrates with active groups, we employed covalent immobilization of un-modified carbohydrates on the SiNW surface by formation of oxime bond through amine groups modified with 3-aminopropyltriethoxysilane, and subsequently aminooxyacetyl groups introduced with Boc-aminooxyacetic acid. Fluorescence microscope was employed to demonstrate the successful surface chemistry for immobilization of carbohydrates on the silicon surface. The label-free electrical detection of carbohydrate-protein interactions was then carried out by the carbohydrate-modified SiNW biosensor. Real-time response of lectin and concanavalin A to galactose and mannose-modified SiNW biosensors, respectively, as function of time was achieved. It was found that 100 fg/ml lectin was detected by the galactose-modified SiNW biosensors, which was the highest sensitivity compared to that developed by other technologies.
Fully synthetic immuno-modulators and tripartite vaccines for cancer Geert-Jan Boons1, Dr, University of Georgia, Complex Carbohydrate Research Center, 315 Riverbend Road, Athens, Ga, United States, 706-5429161, [email protected]
The mucin MUC1 is typically aberrantly glycosylated by epithelial cancer cells manifested by truncated O-linked saccharides. The resultant glycopeptide epitopes can bind cell surface major histocompatibility complex (MHC) molecules and are susceptible to recognition by cytotoxic T-lymphocytes (CTLs), while aberrantly glycosylated MUC1 protein on the tumor cell surface can be bound by antibodies to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). We have identified the minimum requirements to consistently induce CTLs and ADCC-mediating antibodies specific for the tumor form of MUC1 resulting in a therapeutic response in a mouse model of mammary cancer. The vaccine is composed of the immunoadjuvant Pam3CysSK4, a peptide Thelper epitope and an aberrantly glycosylated MUC1 peptide. The vaccine produced CTLs, which recognized both glycosylated and nonglycosylated peptides, whereas a similar nonglycosylated vaccine gave CTLs which recognized only nonglycosylated peptide. Antibodies elicited by the glycosylated tripartite vaccine were significantly more lytic compared to the unglycosylated control.
Structures of carbohydrate-associated epitope on CA215 expressed in ovarian and cervical cancer cell lines and proposed biosynthetic pathways Yin Gao1, Queen's University, Biomedical and Molecular Sciences, 94 Stuart St., Etherington Hall room 2051, Kingston, Ontario, K7L 3N6, Canada, 613-533-6000ext77399, [email protected]
Ovarian and cervical cancers are the most common causes of cancer death from gynecologic tumors in the world. The cancer antigen CA215 was initially identified by RP215, a monoclonal antibody that was originally generated against OC-3-VGH ovarian cancer cells. With RP215 as the probe, CA215 was determined to be a widespread cancer marker and was detected in many different human cancer tissues. Significant elevation of CA215 in serum was found in patients with ovarian and cervical cancers. Since RP215 was shown to react with carbohydrate-associated epitope(s) on CA215, we used highly purified CA215 isolated from both ovarian and cervical cancer cells and carried out O-glycan structural characterization through MALDI-TOF mass spectrometry, lectin-staining and immunohistochemical analysis. The common O-glycan cores 1 to 4 have been observed, as well as mono-fucosylated and di-fucosylated core 1 to 3 based structures. The predominant structures carried by CA215 were sialyl-T, sialyl-Tn and di-sialyl-T antigens. Epitopes such as Lea, Leb, Lex, Ley and SLex were found. Furthermore, the potential pathways for synthesizing CA215 carbohydrate-associated epitope(s) in ovarian and cervical cancer cell lines have been elucidated based on the study of glycosyltransferases involved in O-glycan synthesis. The biosynthetic pathways of mucin type O-glycans in ovarian and cervical cancer cells are similar to a selected number of prostate cancer cells. These results are the foundation for the development of glycosyltransferase targets, effective anti-cancer vaccines and RP215-based anti-cancer drugs in humans.
Antiglycan antibodies as biomarkers for personalized medicine Jeffrey Gildersleeve1, 376 Boyles St, Building 376, Rm 208, Frederick, MD, 21702, United States, 301-846-5699, [email protected]
Cancer vaccines have significant potential as therapeutics to treat cancer, but they typically only provide a clinical benefit in a subset of patients. To optimize their clinical use, there have been major efforts to identify predictive biomarkers (markers that could be used to select patients that are likely to have a positive response) and biomarkers of efficacy (markers that could be used to determine if a patient being treated is having a favorable response). While responses to proteins have been studied at length as potential biomarkers, immune responses to glycans have been largely understudied. Our group uses chemical synthesis and glycan array technology to study the roles of anti-glycan antibodies in the development, progression, and treatment of cancer. Using the array, we have evaluated immune responses induced by a poxvirus-based prostate cancer vaccine (PROSTVAC-V/F) that is currently in Phase III clinical trials. In total, we have profiled over 140 patients from two separate Phase II clinical trials. We demonstrate that pre-existing serum antibodies to blood group A correlate strongly with survival in both patient groups. Moreover, we show that the vaccine induces anti-glycan antibody responses and that these responses have statistically significant correlations with survival. Finally, we provide the first evidence of antigen spreading to glycans induced by a cancer vaccine and demonstrate that antigen spreading correlates with a worse clinical outcome for patients treated with PROSTVAC-V/F. These results highlight the importance of anti-glycan immunity for cancer vaccines and illustrate the utility of anti-glycan antibodies as biomarkers for personalized medicine.
Imaging glycans in the process of apoptosis Peng Wu1, 1301 Morris Park Ave., Bronx, NY, 10461, United States, 718-678-1164, [email protected]
The glycome, the full complement of glycans that a cell produces, is an attractive target for molecular imaging. When a cell undergoes a transition of state, its glycome will change. In this talk, I will discuss our recent progress in imaging glycans in cells undergoing apoptosis.
Chemical tools for disease glycoprofiling Carolyn R. Bertozzi1, Professor, Ph.D., University of California, Berkeley, Department of Chemistry, 820 Latimer, Berkeley, CA, 94720-1460, United States, 510-643-1682, 510-643-2628, [email protected]
Changes in protein and cell-surface glycosylation are hallmarks of many acquired disease states such as cancer and inflammation. Accordingly, the ability to discern glycomic signatures in vitro and in vivo has applications to biomarker discovery and clinical diagnostics. This presentation will focus on chemical tools for probing changes in glycosylation on cells in vivo and proteins obtained from tissue samples. These include metabolic labeling with unnatural sugars, bioorthogonal chemistries for selective sugar tagging, and glycoproteomics platforms for biomarker discovery.
Uncovering a biphasic catalytic mode of C5-epimerase in heparan sulfate biosynthesis Jian Liu1, PhD, University of North Carolina, Eshelman School of Pharmacy, Division of Chemical Biology and Medicinal Chemistry, Chapel Hill, North Carolina, 27599, United States, 919-843-6511, [email protected]
Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C5-epimerase (C5-epi) is a key enzyme in this pathway. C5-epi is known for being a two-way catalytic enzyme, displaying a “reversible” catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C5-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an “irreversible” catalytic mode. Our data found that C5-epi displaying “reversible” or “irreversible” catalytic mode of C5-epi strictly depends on the saccharide substrate structures. The biphasic mode of C5-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.
From chemical glycosylation to expeditious oligosaccharide synthesis Alexei V. Demchenko1, Professor, One University Blvd, St. Louis, MO, United States, 314-516-7995, [email protected]
The involvement of complex carbohydrates in a wide variety of disease-related cellular processes has given this class of natural compounds tremendous diagnostic and therapeutic potential. While scientists have been able to successfully isolate certain classes of natural carbohydrates, the availability of pure natural isolates is still inadequate to address the challenges offered by modern glycoscience. As a consequence, chemical synthesis has become a viable means to obtain both natural complex carbohydrates and unnatural analogues thereof. Unfortunately, chemical synthesis of oligosaccharides of even moderate complexity still remains a considerable challenge, and many more complex structures are not available at all. As such, the development of efficient methods for chemical glycosylation and expeditious oligosaccharide and glycoconjugate synthesis remains a demanding area of research.
At the core of this presentation is the development of new methods, strategies, and technologies for chemical glycosylation and expeditious oligosaccharide assembly. New innovative tools for oligosaccharide synthesis will be discussed in light of recent results. The effectiveness of the methods developed will be illustrated by the synthesis of medicinally relevant oligosaccharides and conjugates thereof. This work was generously supported by awards from the NIGMS and NSF.
Siglec-ligand decorated nanoparticles for induction of B cell tolerance James C. Paulson1, Professor, Ph.D., The Scripps Research Institute, Department of Chemical Physiology, 10550 N. Torrey Pines Road, MEML71, La Jolla, CA, 92037, United States, 858-784-9634, [email protected]
Maintenance of peripheral B cell tolerance to self-antigens is still poorly understood and of high interest with respect to the roles of B cells in autoimmune diseases. The B cell receptor is highly regulated by co-receptors that have the potential to aid in distinguishing between 'self' and 'non-self' and minimize inappropriate activation to self-antigens. B cell siglecs, CD22 and Siglec-G/10, are unique among inhibitory co-receptors by their recognition of sialic acid-containing glycans as 'self'. Previously we showed that multivalent presentation of high affinity CD22 ligands in cis with a T-independent antigen can induce tolerance to B cells (Duong et al., J. Exp. Med., 2010). We have now developed high affinity ligands selective for CD22 and Siglec-G to investigate their respective roles in induction of B cell tolerance. By covalent attachment to lipids, the ligands can be readily incorporated into antigen bearing liposomal nanoparticles for co-presentation to B cells. Using this platform, robust B cell tolerance is achieved towards both T-independent and T-dependent antigens in mice. Mechanistic studies demonstrate that CD22 and Siglec-G act cooperatively to maintain peripheral B cell tolerance by recognition of sialic acid-containing glycans as 'self' that are expressed on all mammlian cells. This platform has potential for antigen specific tolerization of B cells in a therapeutic setting. (NIH grants AI050143, AI099141, CA013889 and HFSP Fellowship LT001099/2010-L)
Applications of nanoporous materials to carbohydrate recognition and synthesis Keith J Stine1, Professor, University of Missouri - Saint Louis, Department of Chemistry and Biochemistry, Center for Nanoscience, One University Boulevard, Saint Louis, MO, 63121, United States, 314-516-5346, 314-516-5342, [email protected]
The expanding field of glycoscience is revealing evermore biologically significant roles for carbohydrate structures in human biology and disease, and leading to advances in both diagnostic and therapeutic applications of carbohydrate structures. Nanoporous materials offer significant opportunities to contribute to the advancement of glycoscience, with applications possible in the fields of supported synthesis, glycoprotein detection, and assay of carbohydrate-protein interactions. Nanoporous gold, with its interconnected network of pores and ligaments typically in the tens of nanometers size range, is a material of high interest for such applications. The material has great versatility and can be prepared as thin supported coatings on the surfaces of electrodes or transducers, or as free-standing monolithic structures through which solutions can flow. Our research to date has demonstrated applications of nanoporous gold in all three of the aforementioned fields. In the area of supported synthesis, we have developed surface-tethered iterative carbohydrate synthesis (STICS). Of special recent interest, is the ability to surface modify monolithic structures of nanoporous gold with self-assembled monolayers that present carbohydrates or can be coupled to lectins. In this presentation, the applications of nanoporous gold to supported synthesis, assay of lectin-carbohydrate binding, and glycoprotein detection are reviewed. Characterization of these systems using microscopy, electrochemistry, and thermal analysis methods is reviewed. This work was generously supported by awards from the NIGMS.
New approaches to the synthesis of bioactive carbohydrates via transition metal catalysis Hien M. Nguyen1, Assistant Professor, PhD, University of Iowa, Department of Chemistry, E457 Chemistry Building, Iowa City, Iowa, 52242, United States, 319-384-1887, [email protected]
Our group has recently developed new approaches for the effective synthesis of 1,2-cis-2-aminosugars, alpha-glycosyl ureas, and alpha-sialosides via transition metal-catalyzed selective glycosylation. These sugars not only make up one of the most important classes of oligosaccharides and glycoconjugates but are also one of the most challenging glycosidic bonds found in nature. In general, obtaining an adequate supply of these sugars from natural sources is exceedingly challenging. The methods that are being developed in our group are broadly applicable and provide products in high yields and with excellent levels of anomeric selectivity. Our transition metal-catalyzed selective glycosylation methods are currently applied to the synthesis of various bioactive carbohydrates such as heparin oligosaccharides, mycothiol, GPI anchors, mucin O-glycans, antibiotic cinodines, and H. pylori oligosaccharides.
Biosynthesis and recognition of furanose-containing glycans from Mycobacterium tuberculosisTodd L. Lowary1, Professor, University of Alberta, Department of Chemistry, 11227 Saskatchewan Drive, Edmonton, Alberta, Canada, 780-492-1861, [email protected]
Glycans containing five-membered furanose rings are present in a range of microbial species. The interaction between furanose-containing glycans and proteins mediates a number of important biological events, but an understanding of these bind events remains poorly defined at the molecular level. Of particular interest to our group is the organism responsible for the disease tuberculosis, Mycobacterium tuberculosis, which produces an array of glycoconjugates containing furanose rings. The talk will focus on work done to probe the interaction between mycobacterial furanose glycans and two families of proteins: antibodies that recognize the cell wall antigen lipoarabinomannan and a glycosyltransferase that is involved in the biosynthesis of arabinogalactan, the largest structural component of the cell wall.
New cancer immunotherapies based on synthetic carbohydrate antigens and metabolic glycoengineering of cancer cells Zhongwu Guo1, Professor, 5101 Cass Ave., Detroit, Michigan, 48202, United States, 313-577-2557, [email protected]
Tumor-associated carbohydrate antigens (TACAs), namely, carbohydrates uniquely or excessively expressed by tumors, are potentially useful targets for the development of cancer immunotherapies. However, most TACAs identified thus far are very poorly immungenic, thu they cannot provoke robust immune responses useful for cancer therapy. To overcome this problem, we have developed a novel therapeutic strategy, which involves metabolic engineering of cell surface sialo-TACAs. For cancer immunotherapy, patients can be innoculated with a vaccine made of a sialo-TACA derivative containing unnatural sialic acid, and after a robust immune response is established, the patients are treated with correspondingly modified mannosamine derivative as biosynthetic precursor of unnatural sialic acid to initiate the expression of the unnatural sialo-TACA analog on cancer cells. Thereafter, the provoked immune system can selectively target and eradicate cancer cells, which bear the unique sialo-TACA derivative. We have prepared a series of unnatural derivatives of GM3 and sTn antigens and observed that N-phenylacetylated GM3 (GM3NPhAc) and sTn (sTnNPhAc) could form effective vaccines, and our studies have also demonstrated that N-phenylacetyl mannosamine (ManNPhAc) is an excellent precursor for cancer cell glycoengineering. Antibodies obtained with GM3NPhAc were found to mediate strong and specific cytotoxicity to ManNPhAc-treated cancer cell without obvious toxicity to ManNPhAc-treated normal cell under the same condition. In vivo studies further showed that vaccination of mice with a GM3NPhAc conjugate followed by treatments with ManNPhAc could effectively inhibit tumor metastasis, proving the concept and the potential of the new immunotherapy. In the process, we have developed a novel type of fully synthetic carbohydrate vaccines consisting of TACA and and demonstrated that they could induce strong IgG immune responses without the use of an adjvant.
Investigating galectin-mediated cross-linking using glycoengineered cells Brian Belardi1, 2338 Channing Way, Apt. 4, Berkeley, California, 94704, United States, 7032018004, [email protected]
Cell surface multimerization of proteins and lipids is a ubiquitous process that has immense regulatory consequences for functional assemblies. The “galectin lattice” model has been invoked to explain a myriad of cellular processes, ranging from adhesion to malignant transformation. This model hinges on multivalent galectin proteins inducing the clustering of glycoproteins. However, to date, galectin-mediated cross-linking of glycoconjugates has not been directly observed on live cells. We describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers functionalized with a lipid group and a fluorophore. After insertion into live cell membranes, the glycopolymers' fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the “galectin lattice” hypothesis in a physiologically relevant context.
Glycosylation engineering of therapeutic antibodies Lai-Xi Wang1, Prof., PhD, 3817 Palmetto Court, Ellicott City, Maryland, 21042, United States, 410-706-4982, [email protected]
Monoclonal antibodies (MAbs) are an important class of therapeutic glycoproteins. Compelling evidence indicates that the fine structures of the conserved N-glycans attached to the Fc domain modulate the effector functions of IgG antibodies, including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and anti-inflammatory activities. However, most therapeutic monoclonal antibodies are produced as mixtures of glycoforms and it is difficult to control glycosylation at a homogeneous status to maximize a specific beneficial function. This presentation describes a chemoenzymatic method that exploits a novel enzymatic deglycosylation-transglycosylation approach to achieve site-specific Fc glycosylation remodeling. It enables the synthesis of specific homogeneous glycoforms of MAbs and intravenous immunoglobulin (IVIG) to enhance ADCC functions or to gain anti-inflammatory activities. The scope and limitation of the method in biotechnology will be discussed.
Production of chiral organic molecules through synthetic biology Peng George Wang1, Prof., PhD, Georgia State University, Center for Diagnostics and Therapeutics and Department of Chemistry, 100 Piedmont Avenue, Atlanta, GA, 30303, United States, 404-413-3591, [email protected]
Synthesis of chiral organic molecules and complex natural products through catalysis of enzymes in vitro has been increasingly becoming versatile and feasible. For example, dihydroxyacetone phosphate (DHAP)-dependent aldolases catalyzed aldol condensation between the three carbon DHAP and an aldehyde has been particularly attractive because the chirality of the two newly generated chiral centers can be chosen and controlled by the choice of the aldolase enzyme. Thus such enzymatic reactions have been used to produce many rare sugars and other useful organic molecules containing multiple hydroxyl groups. Recently we have transferred such enzymatic reactions into E. coli cells by fusing the aldolase catalyzed pathway between DHAP and an external aldehyde with the intrinsic glycolytic pathway which generates DHAP through synthetic biology approaches. A vector containing both an aldolase gene and a phosphatase gene was transformed into the engineered recombinant E. coli system. The reaction product from the aldol condensation between DHAP and the aldehyde was being accumulated in the broth during fermentation of the engineered E. coli. This system realizes the usage of a 3-carbon structure (DHAP) from natural resources (such as glucose or other carbon sources in the fermentation) for chiral organic molecules and importantly the recycle of phosphates in vivo. The efficiency and yield of the system to produce a variety of chiral organic molecules is being improved through system biology approaches.
Recent advances in glycolipidomic profiling of pathogenic processes Jacquelyn Gervay-Hague1, Professor, One Shields Ave., Davis, CA, United States, 530-754-9577, [email protected]
It is increasingly clear that membrane-bound cholesterol plays an important regulatory role in pathogenic processes. For example, cholesterol has been shown to modulate glycolipid conformations, which can either inhibit or facilitate recognition processes involving raft-associated proteins. There is also recent evidence that cholesterol may directly mask glycosphingolipids such as monosialotetrahexosylganglioside (GM1) and thereby protect the carbohydrate from recognition by toxin-derived proteins including cholera toxin. When one considers these findings in the context of the discovery that many pathogenic bacteria produce enzymes that require extrusion of cholesterol from host cells, an intriguing interplay of cholesterol transport between host and pathogen emerges. Recent advances in our laboratory have led to efficient syntheses of host- and pathogen-associated glycolipids to enable studies focused on glycolipidomic profiling and the influence of cholesterol in pathogenic processes.
Synthetic Thomsen-nouveau (Tn) antigen conjugated to zwitterionic polysaccharide PS A1 for an entirely carbohydrate immunogen construct Peter R Andreana1, Prof., University of Toledo, Chemistry, 12881 Nautica Dr, Belleville, MI, 48111, United States, 734-787-5937, [email protected]
Background and Objectives: Aberrant carbohydrates on cancer cell surfaces are important targets for the possible development of effective cancer vaccines. In recent years, a new class of bacterial polysaccharides, characterized by an alternating zwitterionic charge character motif on adjacent monosaccharides, has been shown to stimulate T- and B-cell immune responses effectively. Our goal is to prepare semi-synthetic vaccines containing tumor associated carbohydrate antigen derivatives conjugated to the zwitterionic polysaccharide PS A1. More specifically the aminooxy Thomsen-Nouveau (Tn, a-GalNAc-O-NH2) antigen. With this immunogen in hand, studies of the immune response in mice is a main objective.
Methodology: The C57BL/6 mice for the immune study were split up into three groups all receiving a weekly i.p. injection. The first group received only PBS buffer, the second group received PS A1 accompanied with TiterMax Gold adjuvant, and third the vaccine construct Tn-PS A1 and the above adjuvant. Mice were euthanized each week from both the PS A1 group and Tn-PS A1 group starting one week after the first injection. The collected sera were then analyzed by ELISA, complement dependent cellular cytotoxicity assays (CDC), and SCID mouse studies with human cancer cell lines HCT-15 and MDA-231.
Results: Large increases in antibody titers were observed for both PS A1 and Tn-PS A1 compared to controls. The sera was also shown to contain IgG3 antibodies. Initial CDC assays and SCID mouse studies have so far proven to be positive but further studies are currently ongoing in our lab(s).
Conclusion: The semi-synthetic vaccine Tn-PS A1 can elicit high titer antibodies with specificity towards the Tn antigen. The presence of IgG3 antibodies implies a T-cell dependent immune response. Future studies will include other TACA-PS A1 constructs such as the TF-PS A1 immunogen and subsequent immunological evaluation.
Chemical probes of oligosaccharide assembly and function Laura L. Kiessling1, 1101 University Avenue, Madison, WI, United States, 608-262-0541, [email protected]
Glycans are critical constituents of all organisms, and every cell on Earth wears a glycan coat. With this context, it is perhaps not surprising that the most abundant organic compounds on the planet are carbohydrate polymers. Bacterial polysaccharides can be essential for viability or contribute to virulence and pathogenesis. Mammalian polysaccharides, such as the glycosaminoglycans, are critical signaling components that regulate pluripotency and development. Despite their fundamental importance, little is known about how polysaccharides are built, how their sequences and structures are controlled, and how they function. This seminar will focus on the use of synthetic substrates, inhibitors, ligands, and probes to address unanswered questions regarding the assembly and function of specific polysaccharides.
Biologics through chemical synthesis Samuel Danishefsky1, PhD, 430 East 67th Street, New York, NY, 10065, United States, 212-639-5502, [email protected]
The traditional line of thought in pharma is that so-called "Small Molecules" are the problems of Chemists. By contrast, biologics (oligonucleotides, oligosaccharides, glycoproteins) are seen to be in the purview of biology and biologists. In this lecture we will describe some striking progress in the application of new chemistry to the synthesis of erstwhile biologics.
Biomimetic cellulose nanofiber composites: From stimuli-responsive materials to biomedical implants Stuart J Rowan1, Prof, PhD, Case Western Reserve University, Department of Macromolecular Sci & Eng, 2100 Adelbert Road, Cleveland, Ohio, 44106-7202, United States, 2163684242, [email protected]
Nanocomposites utilizing cellulose nanocrystals (CNCs) are a burgeoning area of research. CNCs are attractive as reinforcing fillers on account of their high tensile stiffness (up to ca. 140 GPa) and relative abundance in nature. They can be obtained from a range of biosources (e.g. cotton, wood, wheat straw or sea creatures known as tunicates) and can vary in aspect ratio (ca. 10 to 100) depending on the biosource. This talk will focus on our recent use of CNCs to create polymer nanocomposites that mimic the architecture and the mechanic adaptability of the sea cucumber dermis and have lead to the development of a range of new stimuli-responsive materials with applications targeted toward biomedical implants.
De novo synthesis of carbohydrate based natural products for medicinal chemistry George O'Doherty1, Prof., Northeastern University, Chemistry and Chemical Biolgy, 360 Huntington Ave, 102 Hurtig Hall, Boston, MA, 02120, United States, 6173734817, United States, [email protected]
The de novo asymmetric syntheses of the glycosylated natural products and analogues from achiral starting materials have been developed. The asymmetry of the carbohydrate portion of these products was installed by means of the Noyori reduction, diastereoselective palladium catalyzed glycosylation and subsequent diastereoselective post glycosylation transformation. This approach has been widely successful in term of the range a structural motifs it has been applied. Our latest synthetic effort along with subsequent biological evaluations will be presented.
General and convergent route for synthesis of diverse glycosylphosphatidylinositols: Applications in diagnostics and vaccines Sebastian Goetze1, Mr., Max Planck Institute of Colloids and Interfaces, Biomolecular Systems, Arnimallee 22, Berlin, Berlin, 14195, Germany, +493083852443, [email protected]
Glycosylphosphatidylinositol (GPI) glycolipids anchor a large number of proteins in the cell membrane of eukaryotic cells. Their conserved pseudopentasaccharide core (H2N(CH2)2OPO3H6Man12Man16Man14 GlcN16myo-Ino1-OPO3H–) can carry additional phosphoethanolamine, saccharide and lipid substituents. Biological studies of GPIs rely on availability of homogeneous samples of these molecules. To address the need for a diverse set of homogeneous GPI structures we have developed a general synthetic route to GPI molecules. Here, we report on the development of this synthetic strategy, and the application of different branched GPI glycans and glycolipids as diagnostic tools and potential vaccines.
Carbohydrate synthesis and the design of immunomodulatory materials Nicola L.B. Pohl1, Professor, PhD, 800 E. Kirkwood Avenue, Bloomington, IN, 47405, United States, 812-855-9043, 812-855-8300, [email protected]
Cells tout diverse carbohydrate coatings that either aid or subvert animal and human immune responses. For this reason, carbohydrate-based materials are promising as biomaterials. Unfortunately, the use of carbohydrate materials from natural sources are complicated by their isolation as microheterogeneous mixtures and sometimes by the low relative abundances of particular structures needed for a specific biological response. This talk will focus on the design and synthesis of carbohydrates, including by automated oligosaccharide synthesis, and on linkers and conjugation strategies for these sugars for their incorporation into polymeric biomaterials.
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