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Protein Engineering, Design and Selection vol. 17 no. 1 pp. 21-27, 2004
© 2004 Oxford University Press

Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications

Tove Olafsen1,2, Chia-wei Cheung3, Paul J. Yazaki3, Lin Li4, Gobalakrishnan Sundaresan1, Sanjiv S. Gambhir1,5, Mark A. Sherman6, Lawrence E. Williams7, John E. Shively4, Andrew A. Raubitschek8 and Anna M. Wu1,3

1Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 700 Westwood Plaza, Los Angeles, CA 90095, 3Division of Molecular Biology, 4Division of Immunology and 6Division of Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010, 5Department of Radiology and Bio-X Program, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, 7Division of Radiology and 8Department of Radioimmunotherapy, City of Hope National Medical Center, 1500 East Duarte Road, Duarte, CA 91010, USA

2 To whom correspondence should be addressed. e-mail: tolafsen{at}mednet.ucla.edu

An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modification of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-specific conjugation approaches can direct modifications to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide specific thiol groups for chemical modification. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disulfide-bonded dimer. This cysteine-modified diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disulfide bond, the Cys-diabody could be chemically modified using a thiol-specific bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modified site-specifically. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.

Received October 15, 2003; accepted October 15, 2003 Edited by Greg Winter


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