title: Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase.
abstract: We have designed and produced a stable bispecific scFv dimer (bisFv) by non-covalent association of two hybrid VH-VL pairs derived from an anti-neuraminidase antibody (NC10) and an anti-glycophorin antibody (1C3). The bisFv dimer was demonstrated to have binding activity to the two respective target antigens and was evaluated as a reagent for rapid whole blood agglutination assays. The bisFv was expressed in the periplasm of Escherichia coli, from a secretion vector which comprised two cistrons in tandem under the control of a single lac promoter, inducible with IPTG. Each cistron encoded one of the hybrid VH-VL pairs, with V domains separated by a linker region encoding the five amino acids, Gly4Ser. The short linker region was designed to prevent association of VH and VL regions of the same molecule and favour the formation of dimers. The protein synthesized from each hybrid scFv cistron was directed to the E. coli periplasm by the inclusion of distinctive signal secretion sequences preceding each hybrid gene; from pel B of Erwinia cartovora and from gene III of fd phage. The bisFv was affinity-purified from culture supernatants via the C-terminal tag epitope FLAG and was shown, by FPLC on a Superose 6 column, to be consistent in size with that of a scFv dimer. The bisFv was stable for more than 4 months at 4 degrees C and was shown by BIAcore analysis to bind to either target antigen, human glycophorin, or tern N9 neuraminidase. Simultaneous binding to both target antigens was demonstrated when a pre-formed bisFv-neuraminidase complex was shown to bind to immobilized glycophorin. In whole blood agglutination assays, the bisFv dimer was able to agglutinate red blood cells when crosslinked with an anti-idiotype antibody (3-2G12) binding to the NC10 combining site, but no agglutination occurred on binding the antigen neuraminidase. These results are a function of the topology of the epitopes on neuraminidase and have implications for the use of relatively rigid bifunctional molecules (as bisFv dimers) to cross link two large membrane-anchored moieties, in this case, red blood cell glycophorin and neuraminidase, an M(r) 190,000 tetramer.
authors.affiliation: CSIRO, Division of Biomolecular Engineering, CRC for Diagnostic Technologies, Parkville, Victoria, Australia.
authors.forename: J L
authors.initials: JL
authors.lastname: Atwell
authors.forename: L A
authors.initials: LA
authors.lastname: Pearce
authors.forename: M
authors.initials: M
authors.lastname: Lah
authors.forename: L C
authors.initials: LC
authors.lastname: Gruen
authors.forename: A A
authors.initials: AA
authors.lastname: Kortt
authors.forename: P J
authors.initials: PJ
authors.lastname: Hudson
mesh_terms: D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D000915:Antibody Affinity; D000941:Antigens / Q000032:analysis / Q000276:immunology; D001666:Binding Sites, Antibody; D015374:Biosensing Techniques; D002850:Chromatography, Gel; D019281:Dimerization; D006021:Glycophorins / Q000276:immunology* / Q000378:metabolism; D006386:Hemagglutination Tests; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D009439:Neuraminidase / Q000276:immunology* / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D018033:Antibodies, Bispecific; D000941:Antigens; D006021:Glycophorins; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D009439:Neuraminidase