Hypervariable Loop Swapping Analysis Reveals Sequence Determinants of Aggregation-Resistant Human 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 aggregation resistance for human V_H domain antibodies that differ only in the sequence of their complementarity determining regions (CDRs), yet possess dramatically different aggregation propensities in a manner not correlated with their folding stabilities. We find the propensity of domain antibodies to aggregate after being transiently unfolded is not a distributed property of the three CDR loops, but can be localized to a single CDR loop. Moreover, we have identified a mutation in this CDR loop that endows the poorly soluble variant with biophysical properties indistinguishable from the aggregation-resistant variant. Importantly, we find that several other charged mutations in this CDR loop, non-CDR loops and the antibody scaffold are incapable of preventing aggregation. We expect that our identification of highly localized positions within CDR loops that govern aggregation will guide the design and selection of antibodies that not only possess high affinity and folding stability, but also extreme resistance to aggregation.