Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library

Structure-guided phage display was used to select for combinations of interface residues for antibody CH3 domains that promote the formation of stable heterodimers. A CH3 “knob” mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of CH3 “hole” mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner CH3 domain. The CH3 knob mutant was fused to a peptide flag and the CH3 hole library was fused to M13 gene III. Phage displaying stable CH3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected CH3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407′ A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual CH3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366′S:L368′A:Y407′V, had a tm of 69.4°C, which is 4.0deg.C higher than that for the designed heterodimer and 11.0deg.C lower than that for the wild-type homodimer. The phage-selected CH3 mutant maintained the preference for forming heterodimers over homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering homodimers for heterodimerization.