title: Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli.
abstract: We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.
authors.affiliation: Genzentrum Universität München, Max-Planck-Institut für Biochemie, Martinsried, FRG.
authors.forename: P
authors.initials: P
authors.lastname: Pack
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D000941:Antigens / Q000276:immunology; D004926:Escherichia coli / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D007074:Immunoglobulin G / Q000737:chemistry; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology; D014461:Ultracentrifugation
chemical_list: D000941:Antigens; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins