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Title: Solution structure of interleukin-1β bound to a potential therapeutic antibody
Author: Wilkinson, Ian Craig
ISNI:       0000 0004 2721 0796
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2009
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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.
Supervisor: Carr, Mark Sponsor: Biotechnology and Biological Sciences Research Council (BBRSC) ; UCB-Celltech
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available