Anti-Le^a monoclonal antibody SPM 522 recognizes an extended Le^a epitope

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