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Expression and characterization of a humanized cocaine-binding antibody
El-Rashdy M Redwan1, Nicholas A Larsen, Bin Zhou
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Biotechnology and Bioengineering
|March 26, 2003
Summary
Researchers humanized a single-chain variable fragment (scFv) of the cocaine-binding monoclonal antibody (mAb) GNC92H2. This humanized antibody retains high affinity for cocaine, reducing potential immunogenicity for therapeutic applications.
Area of Science:
- Immunology
- Biochemistry
- Structural Biology
Background:
- The murine monoclonal antibody (mAb) GNC92H2 exhibits high specificity and affinity for cocaine.
- Previous work determined the crystal structure of a chimeric Fab construct of GNC92H2.
- Humanization of antibodies is crucial for reducing immunogenicity in therapeutic applications.
Purpose of the Study:
- To achieve framework humanization of the GNC92H2 single-chain variable fragment (scFv) without compromising cocaine-binding affinity.
- To engineer a humanized antibody with enhanced therapeutic potential.
Main Methods:
- Sequence comparison of mAb GNC92H2 with human antibody sequences.
- Structure-based design incorporating 49 mutations to humanize the framework.
- Codon optimization for E. coli expression and gene synthesis.
- Fusion to thioredoxin to improve scFv expression in E. coli.
- Competitive ELISA and equilibrium dialysis to assess binding activity.
Main Results:
- Successful framework humanization of the GNC92H2 scFv construct was achieved.
- The humanized scFv maintained comparable binding affinity to cocaine as the original whole IgG.
- Expression of the scFv was significantly enhanced in E. coli through thioredoxin fusion.
- The refolded scFv construct demonstrated soluble and active antibody properties.
Conclusions:
- Framework humanization of mAb GNC92H2 is feasible without loss of cocaine-binding affinity.
- The humanized scFv construct shows promise for therapeutic development by reducing immunogenicity.
- Engineered antibody fragments can be expressed and refolded effectively in E. coli.