The goal of this work is to develop antibody-based reagents which can be used in humane for treatment of cocaine addiction. These reagents would provide protection against the pharmacodynamic action of cocaine by rapidly reducing the concentration of cocaine in plasma, thus minimizing positive reinforcement by cocaine. They would be used in conjunction with other treatment modalities dealing with drug-taking behavior. They would also find use in pharmacological studies of cocaine in animals. Murine monoclonal antibodies will be developed which catalyze the hydrolysis of cocaine to nonpsychoactive products. The experimental approach will involve two hydrolysis reactions-(1) hydrolysis of cocaine to ecgonine methyl ester and (2) hydrolysis of cocaine to benzoylecgonine. Transition state analogs (TSA) far these reactions will be synthesized and coupled to carrier proteins. Mice will be immunized, and hybridoma cell lines secreting anti-TSA antibodies will be cloned by limiting dilution. Monoclonal anti-TSA antibodies will be tested for catalytic activity in vitro. Catalytic antibody genes will be sequenced and properties of catalytic antibodies will be enhanced by site-directed mutagenesis. Catalytic antibodies or antibody fragments with sufficient reactivity in vitro will be tested in a mouse model for their ability to modify the pharmacokinetics and reduce the psychoactive effects of cocaine in vivo. Antibodies or antibody fragments which are active in vivo will be engineered for possible use in humans. The proposed work is multidisciplinary in nature and will be done using conventional techniques of chemistry, immunochemistry, pharmacology, and molecular biology. These include synthesis of transition state analogs and conjugates, immunization of mice and production of hybridomas, testing of hybridoma supernatants by ELISA, purification of high concentrations of monoclonal antibodies from ascites fluids, the use of chemical methods for measuring conversion of substrate to product, in vitro kinetic and in vivo pharmacokinetic procedures, tests of locomotor activity, gene cloning, gene sequencing, production of antibody fragments, and site-specific mutagenesis. A sensitive immuno-PCR assay for haptens will be developed to test for binding and/or catalytic activity of recombinant antibodies and antibody fragments.
