(405c) Engineering High-Affinity, Aggregation-Resistant Domain Antibodies

High-affinity antibodies are critical for numerous detection and therapeutic applications, yet their utility is limited by their variable propensities to aggregate either at low concentrations for antibody fragments or high concentrations for full-length antibodies. Therefore, determining the sequence and structural features that differentiate aggregation-resistant antibodies from aggregation-prone ones is critical to improving their activity. We have investigated the molecular origins of antibody aggregation for human domain antibodies that differ only in the sequence the loops containing their complementarity determining regions (CDRs), yet such antibodies possess dramatically different aggregation propensities in a manner not correlated with their conformational stabilities. We find the propensity of these antibodies to aggregate after being transiently unfolded is not a distributed property of the CDR loops, but can be localized to aggregation hotspots within and near individual CDRs. Moreover, we have identified charged mutations within and adjacent to CDRs that endow poorly soluble variants with biophysical properties of aggregation-resistant antibodies. Guided by these findings for germline domain antibodies, we have sought to engineer a panel of anti-amyloid antibodies that possess hydrophobic CDRs with extreme resistance to aggregation without diminishing their affinity and specificity. We find that the CDR sequences of anti-amyloid antibodies strongly influence their solubility. We are currently investigating the impact of charge mutations adjacent to hydrophobic CDRs within these antibodies with the goal of engineering high-affinity, aggregation-resistant antibodies.