Binding symmetry and surface flexibility mediate antibody self-association

• Published in: mAbs 2019-10, Vol.11 (7), p.1300-1318
• Main Authors: Schrag, Joseph D, Picard, Marie-Ève, Gaudreault, Francis, Gagnon, Louis-Patrick, Baardsnes, Jason, Manenda, Mahder S, Sheff, Joey, Deprez, Christophe, Baptista, Cassio, Hogues, Hervé, Kelly, John F, Purisima, Enrico O, Shi, Rong, Sulea, Traian
• Format: Article
• Language: English
• Subjects: Aggregation; native folding; prediction method; single point mutation; structure-aggregation relationship
• ISSN: 1942-0862; 1942-0870
• DOI: 10.1080/19420862.2019.1632114

Solution stability is an important factor in the optimization of engineered biotherapeutic candidates such as monoclonal antibodies because of its possible effects on manufacturability, pharmacology, efficacy and safety. A detailed atomic understanding of the mechanisms governing self-association of natively folded protein monomers is required to devise predictive tools to guide screening and re-engineering along the drug development pipeline. We investigated pairs of affinity-matured full-size antibodies and observed drastically different propensities to aggregate from variants differing by a single amino-acid. Biophysical testing showed that antigen-binding fragments (Fabs) from the aggregating antibodies also reversibly associated with equilibrium dissociation constants in the low-micromolar range. Crystal structures (PDB accession codes 6MXR, 6MXS, 6MY4, 6MY5) and bottom-up hydrogen-exchange mass spectrometry revealed that Fab self-association occurs in a symmetric mode that involves the antigen complementarity-determining regions. Subtle local conformational changes incurred upon point mutation of monomeric variants foster formation of complementary polar interactions and hydrophobic contacts to generate a dimeric Fab interface. Testing of popular tools generally indicated low reliabilities for predicting the aggregation propensities observed. A structure-aggregation data set is provided here in order to stimulate further improvements of tools for prediction of native aggregation. Incorporation of intermolecular docking, conformational flexibility, and short-range packing interactions may all be necessary features of the ideal algorithm.