L-ASPARAGINASE IS AN IMPORTANT ADDITION TO COMBINATION CHEMOTHERAPY PROTOCOLS FOR CHILDHOOD ACUTE LYMPHOCYTIC LEUKEMIA.

L-ASPARAGINASE IS AN IMPORTANT ADDITION TO COMBINATION CHEMOTHERAPY PROTOCOLS FOR CHILDHOOD ACUTE LYMPHOCYTIC LEUKEMIA. HOWEVER, IMMUNOLOGICAL REACTIONS TO THIS FOREIGN PROTEIN CAN GREATLY LIMIT ITS THERAPEUTIC EFFECTIVENESS. THEREFORE, THE PRODUCTION OF NON-CROSS REACTING L- ASPARAGINASE VARIANTS CAN EXPAND THE TIME AND BROADEN THE WAYS IN WHICH THE ENZYME CAN BE EFFECTIVELY GIVEN. L- ASPARAGINASE ISOLATED FROM ERWINIA CAROTOVORA DOES NOT CROSS-REACT WITH ANTIBODIES PRODUCED BY THE FDA-APPROVED ENZYME ISOLATED FROM ESCHERICHIA COLI AND HAS BEEN PROVEN TOBE CLINICALLY EFFECTIVE FOR PATIENTS WHO ARE ALLERGIC TO THEE. COLI ENZYME. THIS PROPOSAL WILL UTILIZE MODERN MOLECULARGENETIC AND PROTEIN ENGINEERING TECHNIQUES TO PRODUCE AN EXTREMELY COST-EFFECTIVE PRODUCTION STRAIN AND RECOVERY PROCESS FOR THIS IMPORTANT CLINICAL REAGENT. IN ADDITION, THESE TECHNIQUES MAY BE USED TO CREATE A FAMILY OF NON- CROSS-REACTING VARIANTS TO FURTHER EXPAND THE THERAPEUTIC EFFECTIVENESS OF L-ASPARAGINASE. IN PHASE I OF THE WORK, THE ERWINIA L-ASPARAGINASE GENE WILL BE CLONED. USING THIS CLONED GENE, PHASE II WILL INVOLVE (1) OVERPRODUCTION OF THEENZYME BY ALTERING THE TRANSCRIPTION, REGULATION, AND COPY NUMBER OF THE GENE; (2) DEVELOPMENT OF A PRODUCTION STRAIN FOR THE ENZYME BY PLACING THE GENE IS A NONPYROGENIC HOST THAT CAN BE GROWN TO HIGH CELL DENSITY AT LOW COST AND SECRETE THE PROTEIN; (3) IMPROVEMENT OF THE RECOVERY PROCESSFOR THE ENZYME BY ALTERING ITS AMINO ACID SEQUENCE WITH THE ADDITION OF RECEPTORS THAT WILL FACILITATE SEPARATION BY AFFINITY CHROMATOGRAPHY; AND (4) ALTERATION OF THE ANTIGEN CROSS-REACTIVITY OF THE PROTEIN BY MUTAGENESIS OF IMMUNODOMINANT REGIONS.