Inhibition of cocaine binding to the human dopamine transporter by a single chain anti-idiotypic antibody: its cloning, expression, and functional properties.
Conventional drug development for treatment of cocaine addiction is greatly hindered by the extreme difficulty in designing a selective cocaine antagonist. We employed anti-idiotypic (anti-Id) antibodies to generate cocaine antagonists. The purpose of this study was to investigate the feasibility of this alternative approach. Herein, we describe the molecular cloning, bacterial expression, and functional properties of an anti-Id monoclonal antibody (mAb), designated K2-3f, which possesses an internal image of cocaine within its variable regions. The heavy and light chain variable domains of K2-3f were cloned by reverse transcription-polymerase chain reaction (RT-PCR) and a single chain antibody variable fragment (scFv) was assembled for expression in Escherichia coli. The scFv bound to the human dopamine transporter (hDAT) with moderate affinity (K(a)=5.3 x 10(6) M(-1)) and excellent mimicry of the cocaine molecule completely inhibited cocaine binding at a molar concentration closely resembling in vivo conditions while allowing approximately 90% of equimolar dopamine uptake. Our data suggest that the use of anti-Id antibody as a template for generation of a cocaine antagonist is a promising approach well worth pursuing. If this strategy is successful, it could be applied to potential ligand-receptor interactions in the treatment of other diseases.
Ho
Mitchell
M
Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 South Lincoln Avenue, Urbana, IL 61802, USA.
Segre
Mariangela
M
D000595:Amino Acid Sequence; D000888:Antibodies, Anti-Idiotypic / Q000096:biosynthesis / Q000235:genetics*; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003042:Cocaine / Q000276:immunology / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000276:immunology; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000276:immunology / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D011485:Protein Binding / Q000276:immunology; D057127:Single-Chain Antibodies
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D050483:Dopamine Plasma Membrane Transport Proteins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D057127:Single-Chain Antibodies; C055966:immunoglobulin Fv; C477789:mAb K2-3f scFv antibody; D003042:Cocaine