COUPLING PROTEIN DESIGN AND IN-VITRO SELECTION-STRATEGIES - IMPROVINGSPECIFICITY AND AFFINITY OF A DESIGNED BETA-PROTEIN IL-6 ANTAGONIST

The minibody is a designed small beta-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes disp layed on the surface of filamentous phage. The 61 residue molecule con sists of three strands from each of the two beta-sheets of the variabl e domain of immunoglobulins packed face to face, along with the expose d H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on p hage, we were able to select a minibody with micromolar affinity for h uman interleukin-6 that behaves as a selective cytokine antagonist. Th e minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for ''stringent'' selection such a s monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, w e have isolated a minibody with improved antagonistic potency and grea ter specificity Furthermore, using hIL-6 mutants carrying amino acid s ubstitutions in distinct surface sites it was possible to carefully de fine the cytokine region that binds the minibody. (C) 1996 Academic Pr ess Limited