Identification of B cell epitopes enhanced by protein unfolding and aggregation

Timothy Eyes, James Austerberry, Rebecca Dearman, Linus Johannissen, Ian Kimber, Noel Smith, Angela Thistlethwaite, Jeremy Derrick

Research output: Contribution to journalArticlepeer-review


Aggregation of therapeutic proteins is a key factor in the generation of unwanted immunogenicity, and can result in reduced serum half-life, neutralization of function and adverse health effects. There is currently little information regarding how aggregates interact with B-cell receptors or cognate antibodies at the protein sequence level, or whether non-native, aggregate-induced epitopes predominate in these interactions. Using an antibody fragment (single chain antibody variable fragment; scFv) that forms aggregates readily at low temperature, anti-scFv IgG antibody responses were generated by intraperitoneal injection of BALB/c strain mice with monomer or aggregate preparations.Aggregate-specific immunosignatures were identified by oligo-peptide microarray, using overlapping 15mer peptides based on the linear sequence of scFv, printed onto glass slides. IgG antibodies from mice immunized with aggregated scFv preferentially recognized a patch of overlapping peptides. This region mapped to a β-strand located at the interface between the VH and VL domains. Molecular dynamics simulations indicated that the VL domain is less stable than the VH domain, suggesting the interface region between the two domains becomes exposed during partial unfolding of the scFv during aggregate formation. These data are consistent with the hypothesis that epitopes from partially unfolded states are revealed, or are more fully exposed, in the aggregated state,and that this can augment the IgG antibody response. This observation offers the theoretical possibility that epitopes preferentially associated with aggregates can be identified from the anti-drug antibody serum IgG response which may, in turn, lead to better methods for detection of anti-drug antibody responses, and improved design of therapeutic proteins to control immunogenicity.
Original languageEnglish
Pages (from-to)181-189
Number of pages8
JournalMolecular immunology
Early online date11 Dec 2018
Publication statusPublished - Jan 2019

Research Beacons, Institutes and Platforms

  • Lydia Becker Institute
  • Manchester Institute of Biotechnology
  • Manchester Institute for Collaborative Research on Ageing


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