title: Aggregation-resistant domain antibodies engineered with charged mutations near the edges of the complementarity-determining regions.
abstract: Antibodies commonly contain hydrophobic residues within their complementarity-determining regions (CDRs) that mediate binding to target antigens. Unfortunately, hydrophobic CDRs can also promote antibody aggregation, which is especially concerning for therapeutic antibodies due to the immunogenicity of antibody aggregates. Here we investigate how the sequences of CDRs within single-domain (V(H)) antibodies specific for the Alzheimer's amyloid β peptide can be engineered to resist aggregation without reducing binding affinity. We find that domain antibodies containing clusters of hydrophobic residues within their third CDR (CDR3) are prone to aggregate within days at 25°C and minutes above 70°C. However, inserting two or more negatively charged residues at each edge of CDR3 potently suppresses antibody aggregation without altering binding affinity. We also find that inserting charged mutations at one edge of CDR3 (N- or C-terminal) prevents aggregation, but only if such mutations are located at the edge closest to most hydrophobic portion of CDR3. In contrast, charged mutations outside of CDR3 fail to suppress aggregation. Our findings demonstrate that the sequence of CDR loops can be engineered in a systematic manner to improve antibody solubility without altering binding affinity or specificity.
authors: Department of Chemical & Biological Engineering,  Center for Biotechnology & Interdisciplinary Studies,  Rensselaer Polytechnic Institute, Troy, NY 12180,  USA.
authors: Joseph M
authors: JM
authors: Perchiacca
authors: Ali Reza A
authors: AR
authors: Ladiwala
authors: Moumita
authors: M
authors: Bhattacharya
authors: Peter M
authors: PM
authors: Tessier
mesh_terms: D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000276:immunology*; D000915:Antibody Affinity; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D055550:Protein Stability; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D012995:Solubility
chemical_list: D016229:Amyloid beta-Peptides; D022801:Complementarity Determining Regions; D061905:Single-Domain Antibodies