The affinity‐selection of a minibody polypeptide inhibitor of human interleukin‐6

A major challenge in basic and applied biological research is the engineering of small proteins with pre‐determined structures and novel functions. In a limited number of cases, this has been achieved by de novo design. An alternative combinatorial approach is based on the construction of large libraries of random peptides and on methods for the selection of the desired molecules. Here we describe a successful combination of both the rational design and the combinatorial approaches for developing proteins with useful biological functions, in this case the construction of a specific inhibitor of the cytokine human interleukin‐6. In previous work, the ‘minibody’, a 61 residue polypeptide consisting of a beta‐pleated framework and two hypervariable regions, was designed, synthesized and expressed on f1 phage surface. We report the construction of a repertoire of 50 million minibodies displayed on phage in which the hypervariable regions have been randomized. One polypeptide which binds tightly and specifically to human interleukin‐6 was isolated from this collection of minibody mutants. This particular minibody is an effective inhibitor of the cytokine's biological activity. The approach described here could in principle be applied to other molecular targets.