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Expression and characterization of a humanized cocaine‐binding antibody
Author(s) -
Redwan ElRashdy M.,
Larsen Nicholas A.,
Zhou Bin,
Wirsching Peter,
Janda Kim D.,
Wilson Ian A.
Publication year - 2003
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.10598
Subject(s) - monoclonal antibody , antibody , immunogenicity , recombinant dna , microbiology and biotechnology , chemistry , immunoglobulin fab fragments , fusion protein , humanized antibody , gene , biology , computational biology , biochemistry , complementarity determining region , genetics
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 ≈ 200 n M ) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 Å 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. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 612–618, 2003.