Mechanisms of self-association of a human monoclonal antibody CNTO607

Some antibodies have a tendency to self-associate leading to precipitation at relatively low concentrations. CNTO607, a monoclonal antibody, precipitates irrevers-ibly in phosphate-buffered saline at concentrations above 13 mg/ml. Previous mutagenesis work based on the Fab crystal structure pinpointed a three residue fragment in the heavy chain CDR-3, 99FHW100a, as an aggregation epitope that is anchored by two salt bridges. Biophysical characterization of variants reveals that F99 and W100a, but not H100, contribute to the intermolecular inter-action. A K210T/K215T mutant designed to disrupt the charge interactions in the aggregation model yielded an antibody that does not precipitate but forms reversible aggregates. An isotype change from IgG1 to IgG4 pre-vents the antibody from precipitating at low concentra-tion yet the solution viscosity is elevated. To further understand the nature of the antibody self-association, studies on the Fab fragment found high solubility but sig-nificant self- and cross-interactions remain. Dynamic light scattering data provides evidence for higher order Fab structure at increased concentrations. Our results provide direct support for the aggregation model that CNTO607 precipitation results primarily from the specific inter-action of the Fab arms of neighboring antibodies followed by the development of an extensive network of antibodies inducing large-scale aggregation and precipitation