Tumor-associated ganglio-N-triosylceramide. Target for antibody-dependent, avidin-mediated drug killing of tumor cells.

The tumor-associated glycolipid, ganglio-N-triosylceramide (GaMAcS1 + 4GalSl -+ 4GlcPl 3 1Cer; GgOseaCer) was used as a target for antibody-conjugated drug killing of tumor cells. Because of antibody inactivation by drug conjugation, targeting systems have been developed in which administration of biotinyl-anti-glycolipid antibodies was followed by successive addition of: (a) avidin and biotinyl drug (e.g. neoearzinostatin); (b) avidin and biotinyl phospholipid liposomes in which actinomycin D was encapsulated; and (c) drug encapsulated in liposomes covalently linked to avidin. Successful targeting and killing of tumor cells expressing ganglio-N-triosylceramide was observed in System a above. Liposome targeting was successful in Systems b and c but liposome-targeted killing of the tumor cells was not efficient. Anti-ganglio-N-triosylceramide antibodies were derivatized with biotin-N-hydroxysuccinimide with fuIl retention of their antibody function including complement-dependent cytolytic activity. In both Systems a and b above, avidin was used to bridge biotin-substituted drugs or drug carriers to biotinyl antibody bound to cells. Targeting required the presence of biotin on both the antibody and the drug or drug liposomes. Absorption of biotinyl antibody with guinea pig red blood cells (which contain ganglio-N-triosylceramide) abrogated targeting. Furthermore, ganglio-N-triosylceramide, on the target cells was identified chemically as three glycolipid spots separated on thin Iayer cbromatography. These were characterized as having the same carbohydrate but different ceramides. Biotinyl neocarzinostatin was synthesized with decreased biological activity, but it gained the capacity to be directed to cells with avidin. Biotinyl liposomes encapsulating actinomycin D were prepared from biotinylphosphatidylethanolamine, lecithin, and cholesterol. In System c above, avidin was covalently linked to liposomes prepared with sialyllactosylceramide by reductive amination after the gentle periodate oxidation of the liposomes. This technique provides a useful basis for the covalent coupling of liposomes to biologically active proteins in general.