Probing a protein-protein interaction by in vitro evolution.
In this study, we used in vitro protein evolution with ribosome and phage display to optimize the affinity of a human IL-13-neutralizing antibody, a therapeutic candidate for the treatment of asthma, >150-fold to 81 pM by using affinity-driven stringency selections. Simultaneously, the antibody potency to inhibit IL-13-dependent proliferation in a cell-based functional assay increased 345-fold to an IC50 of 229 pM. The panoply of different optimized sequences resulting from complementarity-determining region-targeted mutagenesis and error-prone PCR using ribosome display was contrasted with that of complementarity-determining region-targeted mutagenesis alone using phage display. The data highlight the advantage of the ribosome-display approach in identifying beneficial mutations across the entire sequence space. A comparison of mutation hotspots from in vitro protein evolution to knockout mutations from alanine scanning demonstrated that in vitro evolution selects the most appropriate positions for improvements in potency without mutating any of the key residues within the functional paratope.
Thom
George
G
Cambridge Antibody Technology, Milstein Building, Granta Park, Cambridge CB1 6GH, United Kingdom.
Cockroft
Alexis C
AC
Buchanan
Andrew G
AG
Candotti
Cathy Joberty
CJ
Cohen
E Suzanne
ES
Lowne
David
D
Monk
Phill
P
Shorrock-Hart
Celia P
CP
Jermutus
Lutz
L
Minter
Ralph R
RR