CONSTRUCTION, EXPRESSION, AND ACTIVITY OF A BIVALENT BISPECIFIC SINGLE-CHAIN ANTIBODY

This report describes the design, construction, and expression of a bi valent bispecific single-chain antibody (SCA) protein in Escherichia c oli. The bispecificity of the bivalent protein was based on two previo usly constructed monovalent single-chain antibody molecules possessing distinct specificities, SCA 4-4-20 (anti-fluorescein) and SCA 04-01 ( anti-single-stranded DNA). A flexible linker, modeled after a secreted fungal cellulase protein, was incorporated as the interdomain linker covalently joining the two active sites. Bivalent bispecific SCA prote in that accumulated in bacteria as insoluble inclusion bodies was harv ested, denatured, refolded, and affinity-purified in vitro. Affinity-p urified bivalent bispecific SCA showed nearly identical ligand binding properties at each site relative to the individual monovalent single- chain antibody prototype molecules. In both solid and solution phase b inding assays, the bivalent bispecific single-chain antibody simultane ously bound both ligands (fluorescein and (dT)6). Construction of a mo del bivalent bispecific molecule provides a foundation for future asse mbly of similar molecules designed to identify parameters involved in enhanced binding of antibodies due to avidity and dual specificity.