Abstract
We report the utility of in vitro refolding in the preparation of monomorphous hEx3 bispecific diabodies with epidermal growth factor receptor and CD3 retargeting from insoluble aggregates in Escherichia coli. Appropriate interaction between cognate variable heavy and light chains led to the formation of functional hEx3 heterodimers in a diabody format rather than inactive homodimers. The refolded hEx3 was found to exhibit almost the equivalent activity to the hEx3 and single-chain hEx3 (hEx3-scDb) prepared in a mammalian secretion system. We suggest that the preparation of hEx3 from bacterial insoluble material by means of in vitro refolding would be useful for industrial-scale production of the diabody for its potential use in clinical studies.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Antibodies, Bispecific / chemistry*
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Antibodies, Bispecific / immunology*
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Antibodies, Bispecific / metabolism
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Antibodies, Bispecific / pharmacology
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Antineoplastic Agents / chemistry
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Antineoplastic Agents / immunology
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Antineoplastic Agents / metabolism
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Antineoplastic Agents / pharmacology
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CHO Cells
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Cell Line, Tumor
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Cell Proliferation / drug effects
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Chromatography, Gel
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Cricetinae
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Cricetulus
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Dimerization
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ErbB Receptors / immunology*
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Gene Expression
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Humans
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Immunoglobulin Heavy Chains / metabolism
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Immunoglobulin Light Chains / metabolism
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Immunoglobulin Variable Region / metabolism
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Protein Renaturation
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Protein Structure, Quaternary
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / immunology
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Recombinant Fusion Proteins / metabolism
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Recombinant Fusion Proteins / pharmacology
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Substrate Specificity
Substances
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Antibodies, Bispecific
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Antineoplastic Agents
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Immunoglobulin Heavy Chains
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Immunoglobulin Light Chains
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Immunoglobulin Variable Region
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Recombinant Fusion Proteins
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ErbB Receptors