A Vaccine for Drug Addiction: Anti-Cocaine Catalytic Antibody

by Xiangyi Ivy Wang

A Vaccine for Drug Addiction: Anti-Cocaine Catalytic Antibody Xiangyi Ivy Wang, Hans-Eric Aronson and Donald W. Landry

Department of Medicine
College of Physicians and Surgeons
Columbia University Cocaine is one of the most abused drugs in the United States. Its addiction and overdose are still untreatable. By applying protein engineering approach, we generated a novel class of artificial anti-cocaine catalytic antibody which may serve as an immunizer/vaccine. When giving a cocaine addict a single dose of this vaccine, it will destroy cocaine molecules in the blood circulation before the drug can even get to the brain. The enzyme could remain active and "on duty" in the bloodstream for as long as a year before another injection is needed. In this study, antigens (cocaine transition-state analogies conjugated to Bovine Serum Albumin) were immunized to Balb/C mice and monoclonal antibodies were prepared by standard hybridoma technique. We identified 9 different catalytic hybridoma monoclonal antibodies (Mabs) by cDNA library, DNA and protein sequencing, and subcloning technique. These are IgGs with 4 clones that contain lambda light chain and the other 5 clones contain kappa light chain. Their different Complementarity Determining Regions (CDRs) showed a high immunoglobin diversity. The most active one, Mab 15A10, exhibited a rate acceleration (kcat/kuncat=2.3x104) sufficient to commence preclinical studies. However, due to it's immunoincompatibility (they are all murine Abs, can't be directly used in human), Mabs have limited applications in human being. In order to eliminate the effector function (constant region) of Mabs, we generated a Single Chain Fragment of Variable (ScFv) with an intact antigen (cocaine) binding site. The heavy chain variable region (VH) and the light chain variable region (VL) DNA were generated from Mab 15A10 mRNA by using the degenerated oligonucleotide primers and the polymerase chain reaction (PCR). The carboxyl terminus of VH, and the amino terminus of VL were covalently joined by a flexible polypeptide linker, which consisting of stretches of Glycine and Serine residues. This ScFv was then subcloned into the pCANTAB SE, a phagemid vector. Panning selections were performed against cocaine transition-state analogies on this recombinant phage. Escherichia coli HB2 151 cells were infected to produce a soluble protein, which was further purified by Anti-E tag affinity columns and by High Performance Liquid Chromatography. This purified protein maintained cocaine transition-state analogies affinity as determined by an Enzyme-Linked Immunosorbent Assay (ELISA), and cocaine catalytic activity via its capacity to release titrated benzoic acid from cocaine labeled at the phenyl ring. Hence, we not only successfully generated 9 different clones of catalytic antibodies, but also successfully subcloned and expressed the most active anticocaine catalytic ScFv. Future preclinical and clinical studies will be carried out.