title: Expression and characterization of a humanized cocaine-binding antibody.
abstract: The murine immunoglobulin G (IgG) cocaine-binding monoclonal antibody (mAb), GNC92H2, is notable for its exquisite specificity for cocaine, as opposed to chemically-related cocaine metabolites, and for its moderately high affinity (K(d) approximately 200 nM) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 A resolution. Herein, we report the successful framework humanization of a single-chain Fv (scFv) GNC92H2 construct without loss of affinity for cocaine. In brief, we compared the mAb GNC92H2 sequence to human antibody sequences, and used structure-based design to incorporate mutations (total = 49) that would humanize the framework region without affecting the overall shape of the binding pocket or the key cocaine-contact residues. The codons of the rationally designed sequence were optimized for E. coli expression, and the gene was synthesized by a de novo PCR reaction using 14 overlapping primers. Expression of the scFv construct was significantly improved in E. coli by fusion to thioredoxin. Intriguingly, this construct apparently refolds to form soluble active antibody in the reducing environment of the cytoplasm. Competitive ELISA and equilibrium dialysis demonstrated comparable binding activity between the humanized scFv and the whole IgG. The successful humanization of mAb GNC92H2 should enhance its potential therapeutic value by reducing its overall. immunogenicity.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: El-Rashdy M
authors.initials: el-RM
authors.lastname: Redwan
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Larsen
authors.forename: Bin
authors.initials: B
authors.lastname: Zhou
authors.forename: Peter
authors.initials: P
authors.lastname: Wirsching
authors.forename: Kim D
authors.initials: KD
authors.lastname: Janda
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D003042:Cocaine / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D003042:Cocaine