Crystallographic and Biochemical Analysis of Cocaine-Degrading Antibody 15A10^,

Abstract

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.

A Chimeric Human/Murine Anticocaine Monoclonal Antibody Inhibits the Distribution of Cocaine to the Brain in Mice

“…Furthermore, the antibody affinity for cocaine should also be a major determinant of clinical efficacy. Unfortunately, the affinities of the catalytic mAb for cocaine were reported to be approximately 220 M (Larsen et al, 2004) and 55 to 5240 M (Matsushita et al, 2001), whereas the affinity of the anticocaine mAb GNC92H2 was reported to be 200 nM (Larsen et al, 2001). In contrast, the affinity of 2E2 for cocaine is approximately 4 nM (Paula et al, 2004), which is considerably higher than that of the other anticocaine mAb currently under study.…”

“…In addition to 2E2, Redwan et al (2003) have generated, characterized, and "humanized" the murine anticocaine mAb GNC92H2, the murine version of which has been shown to have in vivo efficacy in rat models of cocaine addiction (Carrera et al, 2001). In addition, two catalytic murine anticocaine mAb that are designed to reduce blood cocaine levels through its hydrolysis have been generated and characterized (Matsushita et al, 2001;Larsen et al, 2004). However, the murine catalytic mAb are likely to be immunogenic in humans.…”

Structure-and-mechanism-based design and discovery of therapeutics for cocaine overdose and addiction

“…4, has been designed, synthesized, and used to successfully elicit anti-cocaine catalytic antibodies. 37 The new catalytic antibodies elicited by using TSA-3 so far are not more active than the previously discovered catalytic antibodies against cocaine. However, it might be interesting to test another possible TSA structure, i.e.…”

Identification and Characterization of Single Chain Anti-cocaine Catalytic Antibodies

“…Previously identified mAb 15A10 was converted to scFv format and mutants were examined; however, only relative kinetic data was reported. 55 Antibodies 3F5 and 3H9 represent novel anticocaine catalytic antibodies. Although they are 89% homologous, antibody 3F5 is twice as active as antibody 3H9.…”