Tetravalent miniantibodies with high avidity assembling in Escherichia coli.

We have designed tetravelent miniantibodies assembling in the periplasm of Escherichia coli. They are based on single-chain Fv fragments, connected via a flexible hinge to an amphipathic helix which tetramerizes the molecule. The amphipathic helix is derived from the coiled coil helix of the transcription factor GCN4, in which all hydrophobic a positions of every heptad repeat have been exchanged to leucine and all d positions to isoleucine. Gel filtration shows tetramer assembly of the miniantibody even at low concentrations. As expected, the functional affinity (avidity) of the tetravalent miniantibody is higher in ELISA and BIAcore measurements than that of the bivalent construct and the gain is dependent on surface epitope density.