Gas-phase unfolding assay rapidly predicts structure-function relationships in engineered antibodies with tuned flexibilities
Human (h)IgG2 monoclonal antibodies (mAbs) are potent agonists due, in part, to their ability to undergo disulfide shuffling within their hinge regions. Herein, we describe a rapid, sensitive, collision-induced unfolding (CIU) assay that possesses a predictive relationship between gas-phase protein unfolding and agonism in hIgG2 variants. Furthermore, our results highlight the significance of hinge engineering in tuning mAb structure-function relationships for the development of future biotherapeutics. The agonistic activity of IgG2 antibodies depends, in part, on the flexibility of their hinge region, which is slow to measure in full-length antibodies. Here, the authors develop a rapid mass spectrometry assay that gauges this flexibility and predicts the agonism of engineered antibody variants.
Authors
- Michael R. Armbruster (ORCID: https://orcid.org/0000-0002-9453-3419)
- Christine A. Penfold (ORCID: https://orcid.org/0000-0001-8332-0592)
- Rosendo Villafuerte-Vega (ORCID: https://orcid.org/0000-0002-3969-7381)
- Tatyana Inzhelevskaya (ORCID: https://orcid.org/0000-0003-1976-9533)
- Hayden Fisher (ORCID: https://orcid.org/0000-0003-0093-0921)
- Isabel G Elliott (ORCID: https://orcid.org/0000-0003-4535-5932)
- Mark Steven Cragg (ORCID: https://orcid.org/0000-0003-2077-089X)
- Brandon T. Ruotolo (ORCID: https://orcid.org/0000-0002-6084-2328)
- Patrick J. Duriez (ORCID: https://orcid.org/0000-0003-1814-2552)
- H. T. Claude Chan
- C. Ian Mockridge
Institutions
- Merck & Co., Inc., Rahway, NJ, USA (United States) (US)
- University of Michigan (US)
- European Synchrotron Radiation Facility (FR)
- University of Southampton (GB)
Publication Details
- Journal
- Nature Communications
- Published
- 2026-06-29
- DOI
- https://doi.org/10.1038/s41467-026-75137-y
- Primary Topic
- Monoclonal and Polyclonal Antibodies Research
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Science Foundation
- Cancer Research UK
- National Institutes of Health
- National Institute of General Medical Sciences