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.

Figure 1

Hydrolysis of the benzoyl ester of cocaine (1). Transition state analog hapten GNL (3).

Figure 2

Alignment of complementarity determining regions. A, alignment of heavy chain CDRs. B, Alignment of light chain CDRs. Black, 3A6 class of antibodies; blue, 3F5 and 3H9; bold, important residues; (*), previously identified antibodies 3A6 and 7A1, included for comparison. Sequences aligned with Vector NTI (Informax).

Figure 3

GNL transition state analog bound in antibody 7A1 active site . Residues TyrH50 and TyrL94 hydrogen bond to the phosphonate oxygen. Adapted from the crystal structure of antibody GNL7A1 complexed with the phosphonate transition state analog GNL (Protein Data Bank Accession code 2AJX, Zhu et al,28).