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Protein Engineering vol. 3 no. 8 pp. 703-708, 1990
© 1990 Oxford University Press

RESEARCH-ARTICLE

Site-specific attachment to recombinant antibodies via introduced surface cysteine residues

Alan Lyons, David J. King1, Raymond J. Owens2, Geoffrey T. Yarranton3, Andrew Millican4, Nigel R. Whittle and John R. Adair

Department of Protein Engineering, Celltech Ltd Slough SL1 4EN, UK 1Department of Protein Biochemistry, Celltech Ltd Slough SL1 4EN, UK 3Department of Molecular Genetics, Celltech Ltd Slough SL1 4EN, UK 4Department of Chemistry, Celltech Ltd Slough SL1 4EN, UK

2To whom correspondence should be addressed

Many diagnostic and therapeutic applications of monoclonal antibodies require the covalent linking of effector or reporter molecules to the immunoglobulin polypeptides. Existing methods generally involve the non-selective modification of amino acid side chains, producing one or more randomly distributed attachment sites. This results in heterogeneous labelling of the antibody molecules and often to a decrease in antigen-binding due to the modification of residues close to the antigen-binding site. We report a novel strategy for site-specifically labelling antibodies through surface cysteine residues. Examination of molecular structures was used to identify amino acids of the CH1 domain of the IgG heavy chain that were accessible to solvent but not to larger molecules. Site-directed mutagenesis was used to substitute cysteine residues at these positions in the heavy chain of a mouse/human chimaeric version of the tumour-binding monoclonal antibody, B72.3. Expression of the modified antibody genes in mammalian cells yielded correctly assembled proteins that had thiol groups in pre-determined positions and showed no loss of antigen-binding activity. One of the mutants was used to demonstrate the site-specific attachment of a radio-iodinated ligand to the chimaeric B72.3 antibody.

Keywords: antibody/site-specific/cysteine/recombinant/radio-labelling

Received March 7, 1990; accepted May 10, 1990.


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