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.programmable one-pot synthesis ͉ glycoarray ͉ glycan epitope ͉ Globo H-truncated sequences T he cell-surface glycosphingolipid Globo H is a member of a family of antigenic carbohydrates that are highly expressed on a range of cancer cell lines, especially breast cancer cells (1-4). Furthermore, it has been established that the serum of breast cancer patients contains high levels of antibodies against the Globo H epitope, and this epitope is also targeted by the monoclonal antibodies MBr1 (5-7) and VK-9 (8). As a result, this hexasaccharide has been the focus of studies aimed at anticancer vaccine development (9-15). Many elegant syntheses of Globo H have been reported (11,(16)(17)(18)(19)(20)(21)(22)(23)(24), including one approach that uses the one-pot programmable oligosaccharide synthesis developed in our laboratory (25). Previously, it has been reported that certain truncated Globo H derivatives can still be effective in binding MBr1 and VK-9 antibodies, which could increase the efficiency of immunogen development for vaccine therapy (8,(26)(27)(28)(29)(30). We set out to further characterize the binding specificities of these and cancer patient antibodies by using carbohydrate microarray analysis.Carbohydrate microarrays allow for the direct characterization of carbohydrate-protein interactions. In addition, the attachment of sugars to surfaces can effectively mimic the presentation of these compounds on the cell membrane. A large factor which is present...

Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer

“…GH A was synthesized by a new programmable one-pot strategy developed by our laboratory (19) and reacted with a p-nitrophenyl ester homobifunctional linker (39,40) to yield the half ester B, which after purification, was incubated with a carrier protein as shown in Scheme 1.…”

“…GH is also expressed weakly in normal tissue but only restricted to apical epithelial cells at lumen borders, a site that seems to be immune-inaccessible (9,(15)(16)(17). Moreover, higher levels of anti-GH Ab were detected in the plasma of breast cancer patients (18)(19)(20). Our previous study also revealed that the expression of some glycans, such as GH and Stage-specific embryonic antigen 3 (SSEA3) (also called Gb5), was observed on breast cancer cells and breast cancer stem cells (BCSCs) (21).…”

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

“…Thus, the microarray may provide a more appropriate and more sensitive system for studying protein-carbohydrate interactions than the traditional solution-phase ELISA analysis. Recently, our group has used Globo H and its truncated analog microarrays to profile the binding specificity of its monoclonal and polyclonal antibodies in a qualitative manner (38). In that preliminary study, we found that the polyclonal antibodies from a breast cancer patient can bind both Globo H and its truncated analog without fucose (Gb5), whereas Mbr1 is specific for Globo H (38).…”

“…Recently, our group has used Globo H and its truncated analog microarrays to profile the binding specificity of its monoclonal and polyclonal antibodies in a qualitative manner (38). In that preliminary study, we found that the polyclonal antibodies from a breast cancer patient can bind both Globo H and its truncated analog without fucose (Gb5), whereas Mbr1 is specific for Globo H (38). The binding specificity studies of monoclonal antibodies and human polyclonal antibodies associated with Globo H have prompted us to further investigate the following questions: (i) Are Globo H and Gb5 differentially expressed in disease stages?…”

Elevated levels of serum antibodies against alpha-1, 6-glucan in patients with systemic lupus erythematosus or rheumatoid arthritis

“…Such natural antibodies may play important roles in host defense against microbial invasion and/or in immunoregulation. Amongst the well-studied anti-glycan natural antibodies in humans are the anti-blood group antibodies (Pal et al, 2004;Dinglasan et al, 2005) and antiGal-α-1,3-Gal antibodies (Galili, 2005;Huang et al, 2006). Other known anti-carbohydrate human natural antibodies include those specific for Gal-β-1,3-GalNac (T antigen) and lactose (Gupta et al, 1996;Galili, 2001).…”