DEVELOPMENT OF IMMUNOTOXINS USING A MUTANT OF EXOTOXIN A

THE GOAL OF THE RESEARCH IS TO DETERMINE WHETHER A NEW MUTANT OF PSEUDOMONAS AERUGINOSA ETA WITH IMPAIRED CELL SURFACE RECEPTOR BINDING ACTIVITY WILL ALLOW THE CONSTRUCTION OF IMMUNOTOXINS WITH SUPERIOR CYTOTOXICITY TO IMMUNOTOXINS CONSTRUCTED WITH RICIN A CHAIN. THE LONG TERM OBJECTIVE OF THIS RESEARCH IS TO IMPROVE THE CYTOTOXIC POTENCY OF IMMUNOTOXINS BY USING A MORE POTENT TOXIN THAN IS CURRENTLY AVAILABLE. THE SPECIFIC AIMS ARE TO DETERMINE OPTIMAL CONDITIONS AND CROSSLINKING REAGENTS FOR THE MODIFICATION AND CONJUGATION OF THE MUTANT EXOTOXIN TO ANTIBODY, AND TO COMPARE DIRECTLY THE SPECIFIC AND NON-SPECIFIC CYTOTOXICITY OF THESE CONJUGATES TO CONJUGATES PREPARED WITH RICIN A CHAIN. IMMUNOTOXINS PREPARED EITHER BY DISULFIDE OR THIOETHER LINKAGE BETWEEN ANTIBODY AND THE MUTANT EXOTOXIN WILL BE PREPARED AND TESTED FOR CYTOTOXICITY TO ESTABLISH OPTIMAL CONJUGATION PARAMETERS. BASED ON THESE FINDINGS, IMMUNOTOXINS WILL BE PREPARED WITH THE MUTANT ETA AND SEVERAL MABS. THE SPECIFIC AND NON-SPECIFIC TOXICITY OF THESE IMMUNOTOXINS AND RICIN A CHAIN IMMUNOTOXINS PREPARED WITH THE SAME ANTIBODIES WILL BE DIRECTLY COMPARED BY TESTING ON IN VITRO TARGET CELL LINES. THE SUCCESSFUL ATTAINMENT OF THE OBJECTIVES OF THIS RESEARCH WILL EVENTUALLY MAKE IMMUNOTOXINS WITH INCREASED CYTOTOXIC POTENCY COMMERCIALLY AVAILABLE BOTH FOR RESEARCH PURPOSES AND FOR THE THERAPY OF HUMAN DISEASE.