Anti-Le monoclonal antibody SPM 522 recognizes an extended Le epitope.

Insights into the differential binding characteristics of anti-Le and anti-LeLe monoclonal antibodies (mAbs) provide information to develop LeLe-based cancer immunotherapeutics while avoiding anti-Le autoimmune reactions. We characterized the epitope recognized by anti-Le mAb SPM 522. We synthesized the Le 6-aminohexyl glycoside and report experimental evidence of a minor conformation in solution. The Le and three other 6-aminohexyl glycosides were conjugated to BSA and titration experiments with SPM 522 show that: 1. SPM 522 binds to LeLe better than to Le; 2. the non-reducing Le galactosyl residue is essential to binding. Competitive ELISA experiments using a panel of tri- to pentasaccharide fragments of LeLe as well as Le analogues indicate that: 1. the Le β-d-galactosyl α hydrophobic patch is crucial to binding; 2. the Le fucosyl residue contributes to binding; 3. the Led-galactosyl residue also contributes to binding. These results indicate that anti-Le mAb SPM 522 recognizes the Le[1,3]-β-d-Gal tetrasaccharide. We propose that a major recognition element is the extended hydrophobic surface defined by the Le-β-d-Gal residue extending to the α faces of the β-d-GlcNAc and β-d-Gal residues.