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, GHkeyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GHrelated 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.carbohydrate vaccine | diphtheria toxin

We report here the development of chemoenzymatic methods for the large-scale synthesis of cancer-associated antigens globopentaose (Gb5), fucosyl-Gb5 (Globo H), and sialyl-Gb5 (SSEA4) by using overexpressed glycosyltransferases coupled with effective regeneration of sugar nucleotides, including UDP-Gal, UDP-GalNAc, GDP-Fuc, and CMP-Neu5Ac. The enzymes used in the synthesis were first identified from different species through comparative studies and then overexpressed in E. coli and isolated for synthesis. These methods provide multigram quantities of products in high yield with only two or three purification steps and are suitable for the evaluation and development of cancer vaccines and therapeutics.

The kinetic mechanism of homoisocitrate dehydrogenase from Saccharomyces cerevisiae was determined using initial velocity studies in the absence and presence of product and dead-end inhibitors in both reaction directions. Data suggest a steady state random kinetic mechanism. The dissociation constant of the Mg-homoisocitrate complex (MgHIc) was estimated as 11 ± 2 mM as measured using Mg 2+ as a shift reagent. Initial velocity data indicate the MgHIc complex is the reactant in the direction of oxidative decarboxylation, while in the reverse reaction direction, the enzyme likely binds uncomplexed Mg 2+ and α-ketoadipate. Curvature is observed in the double reciprocal plots for product inhibition by NADH and the dead-end inhibition by 3-acetylpyridine adenine dinucleotide phosphate when MgHIc is the varied substrate. At low concentrations of MgHIc, the inhibition by both nucleotides is competitive, but as the MgHIc concentration increases the inhibition changes to uncompetitive consistent with a steady state random mechanism with preferred binding of MgHIc before NAD. Release of product is preferred and ordered with respect to CO 2 , α-ketoadipate and NADH. Isocitrate is a slow substrate with a rate of V/E t 216-fold lower than that measured with HIc. In contrast to HIc, the uncomplexed form of isocitrate and Mg 2+ bind to enzyme. The kinetic mechanism in the direction of oxidative decarboxylation of isocitrate, on the basis of initial velocity studies in the absence and presence of dead-end inhibitors, suggests random addition of NAD and isocitrate with Mg 2+ binding before isocitrate in rapid equilibrium, and the mechanism approximates rapid equilibrium random. The K eq for the overall reaction measured directly using the change in NADH as a probe is 0.45 M.Homoisocitrate dehydrogenase (3-carboxy-2-hydroxyadipate dehydrogenase; EC 1.1.1.87) (HIcDH) 1 catalyzes the fourth reaction of the α-aminoadipate pathway (AAA) for lysine synthesis, the NAD-dependent conversion of homoisocitrate to α-ketoadipate (α-Ka) (Scheme 1) (1). Among the 20 common proteinogenic amino acids, lysine is the only one known to have two diverse pathways for its synthesis (2). In bacteria, plants and lower fungi such as phycomycetes or algal fungi, lysine is synthesized via the diaminopimelate pathway, beginning with the phosphorylation of aspartate by aspartokinase. However, it is synthesized via the α- † This work is supported by a grant from the National Institute of Health GM 071417 (to P. F. C. and A. H. W.), and the Grayce B. Kerr Endowment to the University of Oklahoma (to P. F. C.). *Corresponding author: E-mail: pcook@chemdept.chem.ou.edu Tel: 405−325−4581 Fax: 405−325−7182. 1 Abbreviations: HIcDH, homoisocitrate dehydrogenase; AAA, α-aminoadipate pathway; 6-PGDH, 6-phosphogluconate dehydrogenase; ICDH, isocitrate dehydrogenase; IPMDH, 3-isopropylmalate dehydrogenase; TDH, tartrate dehydrogenase; NAD, nicotinamide adenine dinucleotide (the + charge on the nicotinamide ring is omitted for convenience); NADH, reduced nicotinam...

Changes in the expression of cell surface glycan are often associated with malignant metastasis. The expression level may be dramatically enhanced during tumor progression. A highly sensitive assay that is capable of detecting low levels of cancer-associated carbohydrate antigens can be a powerful tool for early diagnosis. In this work, an ultrasensitive glycans array using iron oxide/gold core/shell nanoparticles conjugated with antibodies or proteins is developed. A magnetic field is applied to quickly bring nanoparticle labeled proteins or antibodies from a solution to an array of carbohydrates immobilized on glass slides and to help them to encounter the carbohydrates at very low concentration. The gold shell provides a well established platform for conjugation of biomolecules. Well-defined recognition systems, namely, mannose derivatives (Man1, Man4, and Man9) with a mannose binding lectin (Concanavalin A) and a stage-specific embryonic antigens-3 (SSEA-3) with a monoclonal antibody (anti-SSEA-3) were chosen to establish this detection tool. Array systems were conducted to determine their surface dissociation constant (K(D,surface)) and their binding specificity for qualitative and quantitative analysis of carbohydrate-protein and carbohydrate-antibody interactions. When coupled with a signal amplification method based on nanoparticle-promoted reduction of silver, the sensitivity of an iron oxide/gold core/shell nanoparticle-based assay reached to subattomole level in carbohydrate detection.