(467a) Synergy and Stability in the Treatment of Pertussis: Bispecific Antibody Vs Binary Mixture?

Two potent humanized pertussis toxin neutralizing antibodies, 1B7 and 11E6, bind non-overlapping epitopes and inhibit toxin activity through discrete pathways. These characteristics suggest potential therapeutic synergy between the antibodies, which has since been supported by in vitro neutralization and murine challenge experiments. However, production and purification of two antibodies in parallel is costly and binary antibody mixtures exhibit non-ideal mixing characteristics. Herein we report development of a bispecific 1B7/11E6 antibody as a strategy to retain the attributes of each parent antibody while streamlining production and potentially improving protein stability. The bispecific antibody was made by introducing complementary “knobs-in-holes” mutations [T366Y, Y407T] in the C_H3 interface and transiently expressing each antibody in a separate culture. The bispecific antibody was then generated by combining equimoles of 1B7 and 11E6, followed by a controlled reduction and reoxidation of the hinge disulfide bond. The procedure was monitored by SDS-PAGE. The bispecific preparation was further purified by size-exclusion chromatography and the presence of both binding sites confirmed by ELISA and BIAcore. Synergy of the bispecific was compared to the binary mixture through in vitro CHO cell clustering neutralization assays. Colloidal and conformational stability of the bispecific was assessed by biophysical (size exclusion and dynamic light scattering) and biochemical methods (pertussis toxin binding ELISA) after heat stress, freeze-thaw, and long term storage and compared to the behavior of the binary mixture under similar conditions.