Development of ganglioside-based vaccines for use in active tumor immunotherapy

The overexpresssion of gangliosides in melanoma and several other tumors of neuroectodermal origin makes these molecules attractive targets for the development of tumor immunotherapy. To date, moderate immune response to ganglioside-based conjugates and difficulties associated with ganglioside synthesis and conjugation have hampered the development of a viable cancer vaccine.;This work describes the preparation of ganglioside-based conjugates, their evaluation as cancer vaccines, and the development of a new strategy to enhance their immunogenicity. To efficiently gain access to ganglioside analogues amendable to conjugation, a chemoenzymatic synthesis was developed. A lactoside incorporating a truncated ceramide aglycon functionalized for coupling was synthesized chemically on a gram scale. This truncated lactosyl ceramide avoided the extensive hydrophobicity typical of ceramide-containing compounds allowing for enzymatic elaboration of the glycan chain or production of complex gangliosides centered on this core using metabolically engineered bacteria. The truncated glycolipids obtained by this approach were further modified with fatty acid and sphingosine chains of various lengths to generate gangliosides incorporating different ceramide fragments. These glycolipids were conjugated to a carrier protein (tetanus toxoid) via an adipate-linker and the ability of the conjugates to induce an immune response was evaluated in mice.;Mice immunized with these glycoconjugates responded with high titers of antigen specific antibodies of the IgM, but predominantly of the IgG subclass. Further investigation of the antibody binding paratope by solid-phase assay showed recognition of both the carbohydrate epitope and elements of the ceramide moiety. It was also shown that the nature of the lipid chain as well as the site of conjugation have an influence on the immunogenicity of the constructs. Glycolipids linked to the carrier protein via the fatty acid portion of the molecule were significantly less immunogenic than those incorporating a long fatty acid chain. It can be speculated that the presence of a long fatty acid chain imparts flexibility to the molecule, rendering it more accessible for presentation to the immune system. A novel strategy for augmenting the immunogenicity of ganglioside conjugates in which the antigens are targeted to dendritic cells was also explored. Using this protocol, good titers of ganglioside specific antibodies were obtained with very low concentrations of immunogen. This strategy could allow the development of low-dose vaccines in the future.