title: Mutational analysis of domain antibodies reveals aggregation hotspots within and near the complementarity determining regions.
abstract: High-affinity antibodies are critical for numerous diagnostic and therapeutic applications, yet their utility is limited by their variable propensity 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 V(H) domain antibodies that differ only in the sequence of 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 the first CDR (CDR1). Moreover, we have identified a triad of charged mutations within CDR1 and a single charged mutation adjacent to CDR1 that endow the poorly soluble variant with the desirable biophysical properties of the aggregation-resistant antibody. Importantly, we find that several other charged mutations in CDR1, non-CDR loops and the antibody scaffold are incapable of preventing aggregation. We expect that our identification of aggregation hotspots that govern antibody aggregation within and proximal to CDR loops will guide the design and selection of antibodies that not only possess high affinity and conformational stability, but also extreme resistance to aggregation.
authors: Department of Chemical & Biological Engineering, Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
authors: Joseph M
authors: JM
authors: Perchiacca
authors: Moumita
authors: M
authors: Bhattacharya
authors: Peter M
authors: PM
authors: Tessier
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002162:Camelus / Q000235:genetics; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D012995:Solubility
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains