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Solution Structure of Interleukin-1β Bound To a Potential Therapeutic Antibody

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thesis
posted on 26.10.2012, 12:55 by Ian Craig Wilkinson
The ability of antibodies to bind to target proteins with high specificity makes them an important class of biotherapeutic. The minimal functional form of an antibody, a single chain Fv (scFv), consists of two variable domains joined by a linker. Many therapeutics are based on the larger Fab fragments, which consist of two variable and two constant domains. Detailed structural information for potential therapeutic antibody-target protein complexes could clearly provide valuable insights into their mechanisms of action and mode of binding, however, currently there are very few structures available and none determined by NMR-based methods. To date, the collection of detailed structural information for scFvs has been limited by the formation of domain-swapped dimers. This equilibrium results in the formation of almost 50% dimer at the concentrations required for NMR. However, the work reported here shows that the monomeric form can be stabilised when bound to its target protein, which has enabled NMR-based structural analysis. This has allowed triple resonance data to be collected for the first time for a scFv-target protein complex, enabling over 90% of the backbone resonances to be assigned. Collection of chemical shift perturbation data, intraresidue NOEs and backbone amide RDCs has enabled a homology model for the scFv IC8 to be refined and docked to the structure of its target protein IL-1β. This has produced a reliable and well defined structure for the scFv-IL-1β complex. Evidence has also been obtained for a reorientation of the two variable domains induced by IL-1β binding, which could reflect conformational changes associated with antibody signalling. This study demonstrates that NMR spectroscopy can be used to obtain detailed structural information for antibody-target protein complexes, providing valuable information that could be used in the design and selection of future therapeutic antibodies.

Funding

Biotechnology and Biological Sciences Research Council (BBRSC);UCB-Celltech

History

Supervisor(s)

Carr, Mark

Date of award

01/01/2009

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en

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