Single-chain Fv proteins and their fusion proteins

Abstract

Single-chain Fv (sFv) proteins or single-chain antibodies incorporate a designed linker peptide to bridge isolated immunoglobulin variable domains (V L and V H ) into a single polypeptide. Efficient assembly of sFv genes by polymerase chain reaction, oligonucleotide-directed mutagenesis, and synthetic genetic methods is facilitated by the conserved signal, constant, framework, and linker segments that flank the variable domains. Cytoplasmic or periplasmic expression in Escherichia coli of several sFv proteins has been achieved, and refolding and purification protocols have been reported. Over 15 distinct sFv proteins directed against hapten, protein, receptor, or tumor antigens have been prepared and they retain binding affinities or catalytic properties similar to those of the monoclonal antibodies from which they were derived. Functional expression of sFv proteins on the surface of phage has been achieved and sFv genes may be widely exploited as a convenient genetic unit for antibody engineering. The specific linker sequences used in various sFv proteins have lengths of 14 to 25 residues and have been designed by molecular modeling methods and also by empirical selection of flexible, soluble loops. Because a wide range of linkers have produced fully active sFv proteins, linker obstruction of antigen-binding sites or of domain folding should not be a problem even with widely disparate linkers. The generation of sFv fusion proteins has already proven to be a major application of this technology. Bifunctional proteins with an sFv binding specificity fused to a toxin domain have been shown to act as cell-specific immunotoxins. Many other potential sFv fusion proteins with novel multifunctional affinity and effector characteristics can be produced.