Carbohydrate microarray for profiling the antibodies interacting with Globo H tumor antigen.
ABSTRACT: Understanding the specificity of cell-surface carbohydrates interaction with antibodies and receptors is important for the development of new therapeutics and high-sensitivity diagnostics. This approach is, however, limited to the availability of natural and truncated sequences of the oligosaccharides and the sensitivity of the assay system. Reported here is the synthesis of the cancer antigen Globo H hexasaccharide, an epitope found on the cell surface of breast, prostate, and ovarian cancers, and its truncated sequences by using the programmable one-pot synthesis strategy. The saccharides were then arrayed covalently on glass slides with different density and used for the fluorencense-based binding analysis of two monoclonal antibodies against Globo H and the serum from breast cancer patients, to define the specificity of these antibodies. It was shown that the terminal tetrasaccharide binds the monoclonal antibodies equally well as does the hexasaccharide and the fucose residue is required for effective binding. The serum binds both the defucosylated pentasaccharide and the fucosylated hexasaccharide without a significant difference, perhaps because of the polyclonal nature of the serum or the presence of diverse immune responses to different sugar epitopes at various stages. This method requires very small amounts of materials and is more effective and sensitive than the traditional ELISA method, and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment.
Project description:Pancreatic-type ribonucleases are secretory enzymes that catalyze the cleavage of RNA. Recent efforts have endowed the homologues from cow (RNase A) and human (RNase 1) with toxicity for cancer cells, leading to a clinical trial. The basis for the selective toxicity of ribonuclease variants for cancerous versus noncancerous cells has, however, been unclear. A screen for RNase A ligands in an array of mammalian cell-surface glycans revealed strong affinity for a hexasaccharide, Globo H, that is a tumor-associated antigen and the basis for a vaccine in clinical trials. The affinity of RNase A and RNase 1 for immobilized Globo H is in the low micromolar-high nanomolar range. Moreover, reducing the display of Globo H on the surface of human breast adenocarcinoma cells with a small-molecule inhibitor of biosynthesis or a monoclonal antibody antagonist decreases the toxicity of an RNase 1 variant. Finally, heteronuclear single quantum coherence (HSQC) NMR spectroscopy showed that RNase 1 interacts with Globo H by using residues that are distal from the enzymic active site. The discovery that a systemic human ribonuclease binds to a moiety displayed on human cancer cells links two clinical paradigms and suggests a mechanism for innate resistance to cancer.
Project description:Globo-series glycans are human cell-surface carbohydrates that include stem-cell marker SSEA-4 and cancer-cell antigen Globo H. These two hexasaccharides differ only in their terminal saccharide: N-acetylneuraminic acid in SSEA-4 and L-fucose in Globo H. Herein, we evaluated the affinity of the monoclonal antibodies ?-SSEA-4 and ?-GH for the glycans SSEA-4 and Globo H. Using fluorescence polarization, we find that the two monoclonal antibodies have affinity for their cognate glycan in the low nanomolar range, and have negligible affinity for the non-cognate glycan. Using surface plasmon resonance, we find that each cognate affinity is ?20-fold greater if the glycan is immobilized on a surface rather than free in solution. We conclude that the terminal saccharide plays a dominant role in the ability of monoclonal antibodies to recognize these Globo-series glycans and that the extraordinary specificity of these antibodies supports their use for identifying and sorting stem-cells (?-SSEA-4) and as an agent in cancer immunotherapy (?-GH).
Project description:An improved synthesis of the hexasaccharide MBr1 antigen (globo-H) is reported. Enhanced efficiency in the synthesis was necessary for the scale-up production of globo-H, in order to advance globo-H-based anticancer vaccines to clinical trials. The key features of the improved synthesis include preactivation-based glycosylations and a revised iodosulfonimidation/rearrangement.
Project description: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.
Project description:In recent years, many experiments have been conducted for the production and evaluation of anticancer glycoconjugated vaccines in developed countries and many achievements have been accomplished with Globo H derivatives. In the current experiment, a new chemically designed triplicate version of (Globo H)3-diethylenetriamine pentaacetic acid (DTPA)-KLH antigen was synthesized and characterized. Immunization with (Globo H)3-DTPA-KLH, a hexasaccharide that is a member of a family of antigenic carbohydrates that are highly expressed in various types of cancers conjugated with DTPA and KLH protein, induced a high level of antibody titer along with an elevated level of IL-4 in mice. Treatment of tumors with the collected sera from immunized mice decreased the tumor size in nude mice as well. None of the immunized mice illustrated any sign of tumor growth after injection of MCF-7 cells compared to the control animals. These findings, based on the newly presented structure of the Globo H antigen, lend exciting and promising evidence for clinical advancement in the development of a therapeutic vaccine in the future.
Project description:A derivative of the tumor-associated globo H antigen, a complex hexasaccharide, was synthesized by a convergent and efficient [3+2+1] strategy using various glycosylation methods. All glycosylation reactions afforded good to excellent yields and outstanding stereoselectivity, including the installation of cis ?-linked D-galactose and L-fucose. The longest linear sequence for this synthesis was 11 steps from a galactose derivative 11 to give an overall yield of 2.6%. The synthetic target had a free and reactive amino group at the glycan reducing end, facilitating its conjugation with other molecules for various applications.
Project description:We examined the expression in breast cancer stem cells (BCSCs) of Globo H, a potential tumor-associated antigen for immunotherapy of epithelial cancers including breast cancer. Flow cytometry revealed Globo H expression in 25/41 breast cancer specimens (61.0%). Non-BCSCs from 25/25 and BCSCs from 8/40 (20%) expressed Globo H. We showed the expression of stage-specific embryonic antigen 3 (SSEA3), the pentasaccharide precursor of Globo H, in 31/40 (77.5%) tumors. Non-BCSCs from 29/40 [corrected] and BCSCs from 25/40 (62.5%) expressed SSEA3. Like Globo H, SSEA3 expression in normal tissues was predominately at the secretory borders of epithelium, where access to the immune system is restricted. Immunization of mice with Globo H-KLH and alpha-GalCer induced antibodies reactive with Globo H and SSEA3, suggesting that a Globo H-based vaccine will target tumor cells expressing Globo H or SSEA3. We next sought to reduce Globo H expression by siRNA targeting fucosyltransferase (FUT) 1 and 2, which mediate alpha-1,2 linkage of fucose to SSEA3 to generate Globo H. We showed both genes to be involved in the biosynthesis of Globo H. Moreover, FUT2 expression in BCSCs was significantly lower than in non-BCSCs harvested from a primary human breast cancer in NOD/SCID mouse, whereas FUT1 was slightly lower in BCSCs. Thus, the lower expression of Globo H in BCSCs may be attributed to less FUT2/FUT1, and to reduced SSEA3 in BCSCs compared with non-BCSCs. Our findings provide insight into further development of a Globo H-based vaccine and FUT1/FUT2-targeted therapy for breast cancer.
Project description:Two efficient routes for the rapid assembly of the tumor-associated carbohydrate antigen Globo-H hexasaccharide 2 by a preactivation based iterative one-pot strategy are reported. The first method involves the sequential coupling of four glycosyl building blocks, leading to the desired hexasaccharide in 47% overall yield in one-pot synthesis. Although model studies on constructing the challenging Gal-alpha-(1-4)-Gal linkage in Gb3 trisaccharide yielded the desired alpha linkage almost exclusively, a similar approach to assemble the hexasaccharide led to the formation of a significant amount of beta anomer. As an alternative, the second synthesis utilized three components in one pot with the Gal-alpha-(1-4)-Gal linkage preformed, producing the desired hexasaccharide in a similar overall yield as the four component approach. Both methods demonstrate that oligosaccharides containing alpha and beta linkages within the same molecule can be constructed in one pot via a preactivation based approach with higher glyco-assembly efficiencies than the automated solid-phase synthesis strategy. Furthermore, because glycosylations can be carried out independent of anomeric reactivities of donors, it is not necessary to differentiate anomeric reactivities of building blocks through extensive protective group adjustment for chemoselective glycosylation. This confers great flexibilities in the building block design, allowing matching of the donor with the acceptor, leading to improved overall yield.
Project description:Babesia bovis merozoite apical membrane polypeptide Bv60 was found to be rhoptry associated by immuno-electron microscopy and was redesignated rhoptry-associated protein 1 (RAP-1). The N-terminal 300 amino acids of RAP-1 have a high level of sequence similarity to the same N-terminal region of p58, its homolog from Babesia bigemina. However, the interspecies conserved sequences did not include RAP-1 surface-exposed B-cell epitopes as defined by monoclonal antibodies. Furthermore, neither heterologous B. bigemina immune nor monospecific anti-p58 bovine serum binds to whole RAP-1, indicating that the major B-cell epitopes recognized by these sera are also not encoded by the conserved sequences. Truncated RAP-1, expressed by a subclone encoding the N-terminal 235 amino acids, is weakly bound by antibodies in heterologous sera. A peptide representing the longest conserved amino acid sequence (amino acids 121 to 134) in this region is also weakly bound by antibodies in immune bovine sera, and rabbit antibodies induced by and strongly reactive with the peptide alone fail to bind native or denatured RAP-1. Thus, although the conserved region may contain one or more poorly immunogenic B-cell epitopes, these epitopes are inaccessible to antibody in whole RAP-1. The results indicate that the major immunogenic B-cell epitopes of RAP-1, including surface-accessible epitopes bound by monoclonal antibodies, are distinct from the conserved sequences representing putative functional domains.
Project description:Lectins mediate adhesion of pathogens to host tissues, filling in a key role in the first steps of infection. Belonging to the opportunistic pathogen Burkholderia cenocepacia, BC2L-C is a superlectin with dual carbohydrate specificity, believed to mediate cross-linking between bacteria and host cells. Its C-terminal domain binds to bacterial mannosides while its N-terminal domain (BCL2-CN) recognizes fucosylated human epitopes. BC2L-CN presents a tumor necrosis factor alpha (TNF-?) fold previously unseen in lectins with a novel fucose binding mode. We report, here, the production of a novel recombinant form of BC2L-CN (rBC2L-CN2), which allowed better protein stability and unprecedented co-crystallization with oligosaccharides. Isothermal calorimetry measurements showed no detrimental effect on ligand binding and data were obtained on the binding of Globo H hexasaccharide and l-galactose. Crystal structures of rBC2L-CN2 were solved in complex with two blood group antigens: H-type 1 and H-type 3 (Globo H) by X-ray crystallography. They provide new structural information on the binding site, of importance for the structural-based design of glycodrugs as new antimicrobials with antiadhesive properties.