A NOVEL O-ACETYLATED GANGLIOSIDE DETECTED BY ANTI-GD2 MONOCLONAL-ANTIBODIES

Murine monoclonal antibody (MAb) 3F8 was previously shown to react with disialoganglioside G(D2), but not with G(D3), G(T1b), G(D1b), G(D1a), G(M1), G(M3) and G(M4). However, when the base-treatment step was omitted from the standard neuroblastoma ganglioside extraction procedure, immuno-thin-layer-chromatography (ITLC) using 3F8 and other anti-G(D2) MAbs revealed a new ganglioside band, abbreviated as NG (Rf 0.342) besides G(D2) (Rf 0.183). It migrated below G(D3) (Rf 0.358) on high-performance (HP) TLC plate and its binding to 3F8 on ITLC could be inhibited by rat anti-3F8 idiotypic antibody Idio-2, while the binding of G(D2) to MAb 3F8 was not affected. Immunochemical analysis showed that this new neuroblastoma ganglioside contained alkali-sensitive O-acetylated sialic-acid residues recognized by MAb D1.1. After base treatment, its subsequent identity on ITLC was confirmed to be G(D2). Lactonization of G(D2) yielded 2 major bands, with Rf values (0.401, 0.583) distinct from that of the new ganglioside band. In addition, MAb D1.1 did not bind to any of these G(D2) lactones. Of 15 anti-G(D2) MAbs studied, 13 reacted strongly with the novel ganglioside NG. By ITLC, this NG was found in ganglioside extracts of fresh surgical tumor specimens (4/4 neuroblastomas, 1/1 schwannoma and 1/1 anaplastic astrocytoma), and nude mice/rat xenografts (2/2 neuroblastomas, 2/2 osteogenic sarcomas). These data provided the first evidence that O-acetylated G(D2) is a naturally occurring ganglioside derivative in human tumors and that it could cross-react with most anti-G(D2) antibodies.