Optimal charged mutations in the complementarity-determining regions that prevent domain antibody aggregation are dependent on the antibody scaffold

Therapeutic antibodies need to be highly resistant to aggregation due to the high concentrations required for subcutaneous delivery and the potential immunogenicity of antibody aggregates. Human antibody fragmentsusuch as single-domain antibodies (V-H or V-L)uare typically much less soluble than full-length antibodies. Nevertheless, some aggregation-resistant V-H domains have been discovered that are negatively charged at neutral pH and/or enriched in negatively charged residues within the complementarity-determining regions (CDRs). To better understand how to engineer diverse domain antibodies to resist aggregation, we have investigated the solubilizing activity of positively and negatively charged mutations within hydrophobic CDRs of multiple V-H scaffolds that differ in their net charge. We find that negatively charged mutations inserted near the edges of hydrophobic CDRs are more effective than positively charged ones at inhibiting aggregation for V-H scaffolds that are negatively or near-neutrally charged. In contrast, positively charged CDR mutations prevent aggregation better than negatively charged ones for a V-H scaffold that is highly positively charged. Our findings suggest that the net charge of the antibody scaffold is a key determinant of the optimal CDR mutations for preventing aggregation. We expect that our findings will improve the design of aggregation-resistant antibodies with single- and multidomain scaffolds.