Covalent disulfide-linked anti-CEA diabody allows site-specific conjugation and radiolabeling for tumor targeting applications.
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.
Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095.
Tove
T
Olafsen
Chia-Wei
CW
Cheung
Paul J
PJ
Yazaki
Lin
L
Li
Gobalakrishnan
G
Sundaresan
Sanjiv S
SS
Gambhir
Mark A
MA
Sherman
Lawrence E
LE
Williams
John E
JE
Shively
Andrew A
AA
Raubitschek
Anna M
AM
Wu
D000818:Animals; D000906:Antibodies / Q000737:chemistry; D001483:Base Sequence; D001667:Binding, Competitive; D002272:Carcinoembryonic Antigen / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D051379:Mice; D008819:Mice, Nude; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013438:Sulfhydryl Compounds; D013997:Time Factors; D014055:Tomography, Emission-Computed
D000906:Antibodies; D002272:Carcinoembryonic Antigen; D004220:Disulfides; D010455:Peptides; D013438:Sulfhydryl Compounds; D003545:Cysteine