Recognition of Dimeric Lewis X by Anti-Dimeric Le^x Antibody SH2

The carbohydrate antigen dimeric Lewis X (DimLe^x), which accumulates in colonic and liver adenocarcinomas, is a valuable target to develop anti-cancer therapeutics. Using the native DimLe^x antigen as a vaccine would elicit an autoimmune response against the Le^x antigen found on normal, healthy cells. Thus, we aim to study the immunogenic potential of DimLe^x and search internal epitopes displayed by DimLe^x that remain to be recognized by anti-DimLe^x monoclonal antibodies (mAbs) but no longer possess epitopes recognized by anti-Le^x mAbs. In this context, we attempted to map the epitope recognized by anti-DimLe^x mAb SH2 by titrations and competitive inhibition experiments using oligosaccharide fragments of DimLe^x as well as Le^x analogues. We compare our results with that reported for anti-Le^x mAb SH1 and anti-polymeric Le^x mAbs 1G5F6 and 291-2G3-A. While SH1 recognizes an epitope localized to the non-reducing end Le^x trisaccharide, SH2, 1G5F6, and 291-2G3-A have greater affinity for DimLe^x conjugates than for Le^x conjugates. We show, however, that the Le^x trisaccharide is still an important recognition element for SH2, which (like 1G5F6 and 291-2G3-A) makes contacts with all three sugar units of Le^x. In contrast to mAb SH1, anti-polymeric Le^x mAbs make contact with the GlcNAc acetamido group, suggesting that epitopes extend further from the non-reducing end Le^x. Results with SH2 show that this epitope is only recognized when DimLe^x is presented by glycoconjugates. We have reported that DimLe^x adopts two conformations around the β-d-GlcNAc-(1→3)-d-Gal bond connecting the Le^x trisaccharides. We propose that only one of these conformations is recognized by SH2 and that this conformation is favored when the hexasaccharide is presented as part of a glycoconjugate such as DimLe^x-bovine serum albumin (DimLe^x-BSA). Proper presentation of the oligosaccharide candidate via conjugation to a protein or lipid is essential for the design of an anti-cancer vaccine or immunotherapeutic based on DimLe^x.