Synthesis of Azido-Globo H Analogs for Immunogenicity Evaluation.

Globo H (GH) is a tumor-associated carbohydrate antigen (TACA), and GH conjugations have been evaluated as potential cancer vaccines. However, like all carbohydrate-based vaccines, low immunogenicity is a major issue. Modifications of the TACA increase its immunogenicity, but the systemic modification on GH is challenging and the synthesis is cumbersome. In this study, we synthesized several azido-GH analogs for evaluation, using galactose oxidase to selectively oxidize C6-OH of the terminal galactose or N-acetylgalactosamine on lactose, Gb3, Gb4, and SSEA3 into C6 aldehyde, which was then transformed chemically to the azido group. The azido-derivatives were further glycosylated to azido-GH analogs by glycosyltransferases coupled with sugar nucleotide regeneration. These azido-GH analogs and native GH were conjugated to diphtheria toxoid cross-reactive material CRM197 for vaccination with C34 adjuvant in mice. Glycan array analysis of antisera indicated that the azido-GH glycoconjugate with azide at Gal-C6 of Lac (1-CRM197) elicited the highest antibody response not only to GH, SSEA3, and SSEA4, which share the common SSEA3 epitope, but also to MCF-7 cancer cells, which express these Globo-series glycans.

An Alkynyl-Fucose Halts Hepatoma Cell Migration and Invasion by Inhibiting GDP-Fucose-Synthesizing Enzyme FX, TSTA3.

Fucosylation is a glycan modification critically involved in cancer and inflammation. Although potent fucosylation inhibitors are useful for basic and clinical research, only a few inhibitors have been developed. Here, we focus on a fucose analog with an alkyne group, 6-alkynyl-fucose (6-Alk-Fuc), which is used widely as a detection probe for fucosylated glycans, but is also suggested for use as a fucosylation inhibitor. Our glycan analysis using lectin and mass spectrometry demonstrated that 6-Alk-Fuc is a potent and general inhibitor of cellular fucosylation, with much higher potency than the existing inhibitor, 2-fluoro-fucose (2-F-Fuc). The action mechanism was shown to deplete cellular GDP-Fuc, and the direct target of 6-Alk-Fuc is FX (encoded by TSTA3), the bifunctional GDP-Fuc synthase. We also show that 6-Alk-Fuc halts hepatoma invasion. These results highlight the unappreciated role of 6-Alk-Fuc as a fucosylation inhibitor and its potential use for basic and clinical science.

Immunogenicity study of Globo H analogues with modification at the reducing or nonreducing end of the tumor antigen.

Globo H-based therapeutic cancer vaccines have been tested in clinical trials for the treatment of late stage breast, ovarian, and prostate cancers. In this study, we explored Globo H analogue antigens with an attempt to enhance the antigenic properties in vaccine design. The Globo H analogues with modification at the reducing or nonreducing end were synthesized using chemoenzymatic methods, and these modified Globo H antigens were then conjugated with the carrier protein diphtheria toxoid cross-reactive material (CRM) 197 (DT), and combined with a glycolipid C34 as an adjuvant designed to induce a class switch to form the vaccine candidates. After Balb/c mice injection, the immune response was studied by a glycan array and the results showed that modification at the C-6 position of reducing end glucose of Globo H with the fluoro, azido, or phenyl group elicited IgG antibody response to specifically recognize Globo H (GH) and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) (also called Gb5) and stage-specific embryonic antigen 4 (SSEA4). However, only the modification of Globo H with the azido group at the C-6 position of the nonreducing end fucose could elicit a strong IgG immune response. Moreover, the antibodies induced by these vaccines were shown to recognize GH expressing tumor cells (MCF-7) and mediate the complement-dependent cell cytotoxicity against tumor cells. Our data suggest a new potential approach to cancer vaccine development.

Efficient convergent synthesis of bi-, tri-, and tetra-antennary complex type N-glycans and their HIV-1 antigenicity.

The structural diversity of glycoproteins often comes from post-translational glycosylation with heterogeneous N-glycans. Understanding the complexity of glycans related to various biochemical processes demands a well-defined synthetic sugar library. We report herein a unified convergent strategy for the rapid production of bi-, tri-, and tetra-antennary complex type N-glycans with and without terminal N-acetylneuraminic acid residues connected via the α-2,6 or α-2,3 linkages. Moreover, using sialyltransferases to install sialic acid can minimize synthetic steps through the use of shared intermediates to simplify the complicated procedures associated with conventional sialic acid chemistry. Furthermore, these synthetic complex oligosaccharides were compiled to create a glycan array for the profiling of HIV-1 broadly neutralizing antibodies PG9 and PG16 that were isolated from HIV infected donors. From the study of antibody PG16, we identified potential natural and unnatural glycan ligands, which may facilitate the design of carbohydrate-based immunogens and hasten the HIV vaccine development.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Synthesis and vaccine evaluation of the tumor-associated carbohydrate antigen RM2 from prostate cancer.

We have successfully developed a [1+2+3] one-pot strategy to synthesize the RM2 antigen hexasaccharide that was proposed to be a prostate tumor antigen. The structure of the synthetic product was verified by NMR analysis and antibody binding assay using a glycan microarray. In addition, the synthetic antigen was conjugated to a mutated diphtheria toxin (DT, CRM197) with different copy numbers and adjuvant combinations to form the vaccine candidates. After vaccination in mice, we used glycan microarrays to monitor their immune response, and the results indicated that, when one molecule of DT was incorporated with 4.7 molecules of RM2 on average (DT-RM4.7) and adjuvanted with the glycolipid C34, the combination exhibited the strongest anti-RM2 IgG titer. Moreover, the induced mouse antibodies mediated effective complement-dependent cytotoxicity (CDC) against the prostate cancer cell line LNCap.

Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer.

Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH-keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.

Cancer-associated carbohydrate antigens as potential biomarkers for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies. Therefore, developing the early, high-sensitivity diagnostic biomarkers to prevent HCC is urgently needed. Serum a-fetoprotein (AFP), the clinical biomarker in current use, is elevated in only ~60% of patients with HCC; therefore, identification of additional biomarkers is expected to have a significant impact on public health. In this study, we used glycan microarray analysis to explore the potential diagnostic value of several cancer-associated carbohydrate antigens (CACAs) as biomarkers for HCC. We used glycan microarray analysis with 58 different glycan analogs for quantitative comparison of 593 human serum samples (293 HCC samples; 133 chronic hepatitis B virus (HBV) infection samples, 134 chronic hepatitis C virus (HCV) infection samples, and 33 healthy donor samples) to explore the diagnostic possibility of serum antibody changes as biomarkers for HCC. Serum concentrations of anti-disialosyl galactosyl globoside (DSGG), anti-fucosyl GM1 and anti-Gb2 were significantly higher in patients with HCC than in chronic HBV infection individuals not in chronic HCV infection patients. Overall, in our study population, the biomarker candidates DSGG, fucosyl GM1 and Gb2 of CACAs achieved better predictive sensitivity than AFP. We identified potential biomarkers suitable for early detection of HCC. Glycan microarray analysis provides a powerful tool for high-sensitivity and high-throughput detection of serum antibodies against CACAs, which may be valuable serum biomarkers for the early detection of persons at high risk for HCC.

HA-pseudotyped retroviral vectors for influenza antagonist screening.

Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in alpha-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus-mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses.

Glycan microarray of Globo H and related structures for quantitative analysis of breast cancer.

Cancer-associated carbohydrate antigens are often found on the surface of cancer cells. Understanding their roles in cancer progression will lead to the development of new therapeutics and high-sensitivity diagnostics for cancers. Globo H is a member of this family, which is highly expressed on breast cancer cells. Here, we report the development of a glycan microarray of Globo H and its analogs for measurement of the dissociation constants on surface (K(D,surf)) with three different monoclonal antibodies (VK-9, Mbr1, and anti-SSEA-3), to deduce their binding specificity. The glycan microarray was also used to detect the amount of antibodies present in the plasma of breast cancer patients and normal blood donors. It was shown that the amount of antibodies against Globo H from breast cancer patients were significantly higher than normal blood donors, providing a new tool for possible breast cancer diagnosis. Compared with the traditional ELISA method, this array method required only atto-mole amounts of materials and is more effective and more sensitive (5 orders of magnitude). The glycan microarray thus provides a new platform for use to monitor the immune response to carbohydrate epitopes after vaccine therapy or during the course of cancer progression.

Desorption ionization of biomolecules on metals.

Direct desorption ionization of various types of biomolecules on metal substrates without the need of matrices was observed by a time-of-flight mass spectrometer. It provides a new convenient method for detection of small biomolecules without the confusion of ion peaks from matrix compounds. Simple commercial Al foil can be used as the substrate to obtain mass spectra of biomolecules without the need of an etching process to produce a porous surface such as with direct ionization on silicon (DIOS). The desorption and ionization mechanism is also discussed.

Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli.

D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12,800 and 82,900 Da. Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process. In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-beta-D-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm. Lys629 is responsible for the binding of PLP. The apparent K(d) for coenzyme B(6) binding to d-ornithine aminomutase is 224 +/- 41 nm as measured by equilibrium dialysis. The mutant protein, OraSE-K629M, is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E. coli, in vitro refolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation.