Tumor Associated Carbohydrate Antigens (TACAs) are valuable targets for the development of anti-cancer therapeutics. Thus, epitope mapping has become crucial in both vaccine and monoclonal antibody (mAb)-based immunotherapy development. We are using a combination of synthetic carbohydrate chemistry, molecular modeling, and binding experiments to elucidate the epitopes displayed by the TACA dimeric Lewis X (Chart 1). We have reported that, while the two LeX trisaccharides were rather rigid, the β-GlcNAc-(1,3)-Gal linkage that connects the two Lewis X (LeX) trisaccharides (Chart 1, in red) was highly flexible. Indeed, it appears that the hexasaccharide antigen could adopt two distinct conformations in solution and thus, display two distinct families of epitopes.
Our recent work has focused on the characterization of the epitopes recognized by two mAbs: 1. MAb IG5F6 was raised by E. Altman against H. pylori O:3 cells and selected using the H. pylori LPS known to express LeX and LeY antigens.; 2. MAb SH2 was raised by S.-I. Hakomori against dimLeX-ceramide coated on S. Minnesota and shown to bind preferentially polymeric LeX structures.
Titrations using our glycoconjugates LeX-BSA and dimLeX-BSA suggested, as reported previously,[4-5] that both mAbs had greater affinity for dimLeX than for LeX. Competitive inhibition ELISA studies were performed using the dimLeX-BSA conjugate as immobilized antigen and a panel of tri- to hexa- saccharides (including dimLeXOPr) as soluble inhibitors. Much to our surprised, the trisaccharide LeX-OMe was the strongest competitor amongst the panel of oligosaccharides (Figure 1A). In contrast, inhibitions studies performed using LeX-BSA and dimLeX-BSA as soluble inhibitors showed that when presented on BSA, the dimeric Lewis X hexasaccharide was a stronger competitor than the Lewis X trisaccharide (Figure 1B). These results suggest that epitope presentation is driven by the presence of the carrier protein. We propose that the carrier protein favors one of the two possible conformations of dimLeX that displays the epitopes recognized by mAbs IG5F6 and SH2.
- Hakomori, S., Cancer Res. 1985, 45 (6), 2405–2414.
- (a) Wang, J.-W.; Asnani, A.; Auzanneau, F.-I., Bioorg. Med. Chem. 2010, 18, 71747185; (b) Hendel, J. L.; Auzanneau, F.-I., Eur. J. Org. Chem. 2011, (34), 6864‒6876.
- Jackson, T. A.; Robertson, V.; Auzanneau, F. I., J. Med. Chem. 2014, 57 (3), 817–827.
- Altman, E.; Harrison, B. A.; Hirama, T.; Chandan, V.; To, R.; MacKenzie, R., Biochemistry and Cell Biology 2005, 83 (5), 589–596.
- Singhal, A. K.; Ørntoft, T. F.; Nudelman, E.; Nance, S.; Schibig, L.; Stroud, M. R.; Clausen, H.; Hakomori, S., Cancer Res. 1990, 50 (5), 1375–1380.