Synthetic Analogs of Ganglioside GD2 for the Study of Its Cluster and Multivalent Interactions with Antibodies

Abstract Objective: Ganglioside GD2 is a tumor-associated antigen, and several monoclonal anti-GD2 antibodies are currently used in the treatment of high-risk pediatric neuroblastoma. The development of new therapeutics of this class requires a detailed understanding of the mechanisms underlying the interaction between ganglioside GD2 and antibodies, including the influence of the membrane environment. Methods: These factors include cluster (binding of one Fab fragment of an antibody to several ganglioside molecules) and multivalent (binding of different Fab fragments of one antibody to several ganglioside molecules) interactions. To investigate the possibility of such interactions, two analogs of ganglioside GD2 containing several glycan fragments were synthesized, and their binding to anti-GD2 antibodies was evaluated by flow cytometry. Results and Discussion: Two synthetic glycolipids, (GD2)2-D-Ad-DOPE and (GD2)3-T17-DOPE, containing two and three GD2 carbohydrate moieties, respectively, were successfully synthesized. When incorporated into the cell membrane, both constructs exhibited significantly higher binding to anti-GD2 antibodies compared to their monovalent analogs. The (GD2)2-D-Ad-DOPE construct, featuring a “tight” glycocluster that models ganglioside clustering, showed the most pronounced binding, significantly exceeding that of its monovalent counterpart. The (GD2)3-T17-DOPE construct also exhibited increased binding, attributed to a multivalent interaction effect. These findings indicate that anti-GD2 antibodies preferentially recognize clustered GD2 molecules rather than individual gangliosides. Conclusions: The obtained results support the hypothesis that anti-GD2 antibodies target dynamic clusters of gangliosides within membrane rafts rather than individual molecules. The cluster effect of antibody recognition was shown to be more pronounced than the multivalent effect. This study opens new perspectives in the search for complex targets for anticancer therapeutics.