Antibody VH domains as small recognition units.

To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH. Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface. These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners. The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding. VH clones specific for hapten and protein antigens were isolated. Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection. Affinities of soluble VH for hapten were between 100 nM and 400 nM. The VH domains were highly specific, stable and well expressed in Escherichia coli. These positive biophysical properties and their small size make them attractive for biotechnological applications.