HDX-MS Detects Steric Protection and Trimeric Compaction of TNFα after It Binds Receptors or Antibodies

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with autoimmune disorders such as Crohn's disease, rheumatoid arthritis, and psoriasis. Biotherapeutics represent a rapidly growing part of the treatments used to address autoimmune conditions. Therapeutic monoclonal antibodies (mAbs) are designed to neutralize TNFα but exhibit various efficacies. These variations suggest subtle differences in mAbs binding or neutralization mechanisms. TNFα can bind two receptors, TNFR1 and TNFR2. Therapeutic mAbs bind to TNFα and inhibit signaling through both receptors. X-ray crystallography suggests that when mAbs bind to TNFα, neutralization occurs through steric hindrance. In this report, we used hydrogen/deuterium exchange mass spectrometry (HDX-MS) to analyze TNFα bound to three mAbs. Our results revealed a significant allosteric reduction in TNFα dynamic motion after binding mAbs and receptors. These findings provide an improved model for therapeutic mAbs binding and neutralization of TNFα that incorporates both steric and allosteric mechanisms. Using dynamic structural biology techniques such as HDX-MS to identify new details about neutralizing mAbs, TNFR1, and TNFR2 binding to TNFα is an important advance for biotherapeutic development. Understanding and improving signal neutralization mechanisms may ultimately help minimize symptoms in individuals with autoimmune disorders.