Bioengineering of surface GD3 ganglioside for immunotargeting human melanoma cells

N-Propionyl, N-butyryl (N-Bu), and N-benzoyl mannosamine, as precursors of sialic acid biosynthesis, were incubated with human melanoma SK-MEL-28 cells and resulted in the replacement of N-acetyl groups on the cell surface sialic acid residues, including those associated with GD3. Meanwhile, vaccines containing GD3 and modified GD3 tetrasaccharide-keyhole limpet hemocyanin conjugates were synthesized, and BALB/c mice were immunized with them together with monophosphoryl lipid A adjuvant. The GD3Bu-keyhole limpet hemocyanin conjugate raised the highest IgG titers without any cross-reactivity to unmodified GD3. Expression of GD3Bu epitopes on the surface of SK-MEL-28 cells was confirmed in vitro and in vivo by the binding of a polyclonal antiserum and monoclonal antibody (mAb) 2A, both of which specifically recognize GD3Bu, and by mass spectroscopic analysis of glycolipids extracted from cells. Following expression of GD3Bu on the surface of SK-MEL-28 cells, the cells could be lysed by mAb 2A and GD3Bu antiserum in the presence of complement. Although less effective in the control of existing large size tumors ( approximately 10 mm inner diameter) on BALB/c nu/nu mice, mAb 2A in combination with ManNBu effectively protected mice from SK-MEL-28 tumor grafting. This approach may provide a method to augment the immunogenicity of sialylated human antigens and to avoid generating an autoimmune response to them at same time.