Microbiology 04-4 and Pathology R-1, Miami, FL. USA.Successful treatment of neoplastic disease has been impeded by the lack of therapeutic agents which specifically destroy tumor cells. Enzymes which selectively deplete substrates required by tumor cells, but not by normal tissue, could improve therapeutic indices dramatically. Presently, microbial asparaginases are used clinically for treating acute lymphocytic leukemia. While these enzymes should destroy neoplastic cells and spare normal tissues, their use is accompanied by many toxic effects and immunosuppression. The administration of Escherichia colior Erwinia carotovora asparaginase depletes circulating glutamine as well as asparagine. It has been suggested that this glutaminase activity may be responsible for the observed toxicities. We have isolated a glutaminase-free asparaginase from Vibrio succinogenes with potent antilymphoma activity. Previously, we demonstrated that administration of Vibrio asparaginase does not cause the immunosuppression of humoral or cell-mediated responses assaciated with treatment by other microbial enzymes. In the present communication we have evaluated the hepatotoxic effects of different asparaginases since liver damage is the major toxicity associated with treatment by these microbial enzymes. BALBk mice treated with 50 IU of E. coli asparaginase daily for 4 days exhibited diffuse microfatty infiltration within hepatocytes throughout the liver. Cross-sections of liver from V. succinogenes asparaginase-treated mice appeared normal as compared to specimens from control animals. Quantitation of the total amount of extractable lipid from the livers of E. coli asparaginase-treated animals indicated a 45 % and I27 % increase in lipid concentration as compared to controls after 4 and 5 days of treatment, respectively. The Vibrio enzyme did not cause a change in extractable lipid concentration as compared to control animals. Plasma antithrombin 111 activity and albumin, triglyceride and cholesterol concentrations all decreased in E. coliasparaginase-treated mice, confirming hepatotoxicity. No modifications in plasma proteins were observed in mice treated with asparaginase from V. succinogenes. The plasma lipids did decrease minimally but only the levels of cholesterol were shown to be statistically significant from those of controls. The data strongly support the concept that specific asparagine depletion is not significantly hepatotoxic. More importantly, the asparaginase from V. succinogenes may serve as a potent antileukemic agent without causing damage to normal tissues.

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Asparaginases isolated from Escherichia coli and Erwinia carotovora are effective in the treatment of acute lymphoblastic leukemia in man. During treatment with either of these enzymes, patienk frequently experience pronounced toxicity, including liver and pancreatic dysfunction and immunosuppression. The capabilities of these enzymes t o hydrolyze Lglutamine, an important amino acid in mammalian intermediary metabolism, have led investigators to suggest that the glutaminase activity may be responsible for the observed toxicities. Unlike other asparaginases, an enzyme isolated in our laboratory from Vibrio succinogenes, has been shown to be a potent antilymphoma agent and to be highly specific for Lasparaghe. Our previous work has established that the glutaminase-free asparaginase from V. succinogenes does not suppress the in vivo humoral or cell-mediated immune responses of mice to sheep red blood cells, whereas the E. colienzyme abolishes these responses. In this study, we describe the mechanisms by which E. coli asparaginase suppresses specific antibodydependent cell-mediated cytotoxicity. An analysis of the kinetics of appearance of the specific cell-mediated cytotoxic response of spleen cells after immunization with sheep red blood cells revealed that it paralleled the IgG plaque-forming cell response. In addition, 18-h culture rupernatank from immune spleen cells conferred upon non-immune cells the ability t o specifically lyre sheep red blood cells. Pretreatment of immune supernatank with protein A-Sepharose 4 8 absorbed out the soluble arming factor. An analysis of E. coli asparaginase-induced suppression of specific antibody-dependent cellmediated cytotoxicity revealed that a decreased synthesis of specific IgG was responsible for the observed reduction in cytotoxic reactivity. When culture supernatank from spleen cells of immunized animals not treated with asparaginase were added to spleen cells from E. coli asparaginase-treated mice, however, there was an increased cytotoxic response. This suggested that there was an increase in the number or ratio of effector cells for the specific antibody-dependent cell-mediated cytotoxic response in spleens of enzyme-treated animals. Through the characterization of the immunosuppressive effeck of two asparaginases-one having the catalytic capability to hydrolyze L-glutamine and the other lacking this catalytic activity-we now have evidence that the immunosuppressive effects of E. coliasparaginase on specific antibody-dependent cell-mediated cytotoxicity cannot be the result of asparagine depletion alone. Our data demonstrate that the glutaminase-free asparaginase from V. succinogenes is not immunosuppressive and suggest that an asparaginase with high substrate specificity for Lasparagine may be a safer treatment for leukemia patienk.The immunosuppressive effects of antileukemic asparaginases isolated from Escherichia coli and Erwinia carotovora are well established (Crowther, 1971; Schwartz, 1969) but the molecular basis for this immunosuppression is not understood...

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