Identification and Characterization of Single Chain Anti-cocaine Catalytic Antibodies

Abstract

Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis.

A Cocaine Hydrolase Engineered from Human Butyrylcholinesterase Selectively Blocks Cocaine Toxicity and Reinstatement of Drug Seeking in Rats

“…It is therefore conceivable that anti-cocaine antibodies, administered directly or elicited by vaccination, will play some role in future therapy of cocaine abuse. Nonetheless, although efforts to produce more catalytically efficient antibodies continue (McKenzie et al, 2007), true enzymatic CocHs represent a much more promising option.…”

Accelerating Cocaine Metabolism as an Approach to the Treatment of Cocaine Abuse and Toxicity

“…These studies have used other TSAs, but, so far, the catalytic antibodies produced have had K m values in the millimolar range and/or K cat values well below 1 min −1 [37–40]. Attempts to mutate previously identified cocaine catalytic antibodies have not been any more successful [41]. One exception to this is the catalytic antibodies developed by Cashman et al [42].…”

Pharmacokinetic Strategies for Treatment of Drug Overdose and Addiction

“…A variety of anticocaine catalytic antibodies have been generated [70,71], with catalytic properties less favorable than those of BChE mutants or bacterial cocaine esterase. However, pretreatment with one of the most efficient, mAb 15A10, dose-dependently protects rodents against the increased blood pressure, seizures, and death produced by a cocaine challenge and significantly shifts the dose–response curve for iv.…”