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Immunotoxins Containing Glucose Oxidase and Lactoperoxidase with Tumoricidal Properties: In Vitro Killing Effectiveness in a Mouse Plasmacytoma Cell Model¹

Laboratoire d'Immunologie, UPR 6, Groupe de Laboratories de l'Institut de Recherches Scientifiques sur le Cancer, Centre National de la Recherche Scientifique, 94802 Villejuif, France

We have tested the tumoricidal potency of enzyme immunotoxins constructed of antibodies conjugated to glucose oxidase and to lactoperoxidase. Murine plasmacytoma cells were targeted in vitro with the use of affinity-purified rabbit anti-plasmacytoma membrane antibodies (conjugated to glucose oxidase or lactoperoxidase) or rabbit serum raised against plasmacytoma microsome membranes followed by goat antirabbit immunoglobulin conjugates (to glucose oxidase or lactoperoxidase). Cytotoxicity was generated subsequently by incubation of the washed cells in a medium supplemented with glucose and sodium iodide, which were the substrates of these enzymes. This resulted in the presumed metabolic release of highly toxic reduced oxygen species and iodinated derivatives. Targeting of tumor cells with both conjugates, as opposed to one of them alone, produced a synergistic killing effect. The gain of specific versus unspecific cytotoxicity was upwards of 10,000-fold. The killing rates were elevated (t₁₀ values <30 min) and linear over time. The resultant reduction in tumor cell viability was in the order of 5 to 6 logs after only 20 to 90 min of incubation in the glucose/NaI medium. Cytotoxicity was enhanced by the -glutamyl cysteine synthetase inhibitor buthionine-S,R-sulfoximine and by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, while catalase was inhibitory. The results suggest that these enzyme immunotoxins may be suitable for the ex vivo purging of autologous bone marrow grafts.

¹ Supported by Grant 6407 from the Association pour la Recherche sur le Cancer, and Grant 86-T-0739 from the Ministère de la Recherche et de l'Enseignement Supérieur.

² To whom requests for reprints should be addressed, at Laboratoire d'Immunologie, Groupe de Laboratoires de l'IRSC, BP 8, 94802 Villejuif Cedex, France.

³ Present address: Department of Periodontology and Endodontology, Osaka University Faculty of Dentistry, Osaka, Japan.

Received 3/ 8/89. Revised 7/ 6/89. Accepted 7/17/89.