Coupling protein design and in vitro selection strategies: improving specificity 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 displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two beta-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The 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 as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.
Martin
F
F
Department of Biotechnology IRBM P. Angeletti, Pomezia (Rome), Italy.
Toniatti
C
C
Salvati
A L
AL
Ciliberto
G
G
Cortese
R
R
Sollazzo
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017422:Sequence Analysis, DNA; D000093242:Interleukin-6 Inhibitors
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D007136:Immunoglobulins; D015850:Interleukin-6; D000093242:Interleukin-6 Inhibitors; C081296:Minibody (WT)