CProsser_PhDThesis.pdf (76.44 MB)
Probing Antigen Binding Characteristics Using Single Domain Antibodies
thesisposted on 2018-07-10, 15:55 authored by Christine Elaine Prosser
The variable heavy domains of heavy chain antibodies (VHH) possess unique characteristics which make them an attractive alternative to conventional antibody fragments for detailed NMRP based structural analysis. The small size offers an advantage over Fabs and the enhanced stability compared to scFvs is also beneficial. In addition, the elongated complementarity determining regions (CDRs) associated with VHH fragments may facilitate the characterisation of a more diverse range of antibody-antigen interactions as well as providing templates for studying the motion of CDRs. The work described in this thesis illustrates the feasibility of using VHH fragments to gain structural insight into antibody-antigen binding. The successful generation of isotopically labelled protein allowed the acquisition of high quality NMR spectra from which the solution structures of two free VHH fragments were determined. The solution structures revealed significant differences in the conformational dynamics at the antigen-binding site with both flexible and structured CDR loops being identified. The binding sites of both the VHH and the antigen were mapped by NMR minimal shift analysis. The work discussed in the latter chapters focuses on the structure of VHH 67, its interactions with ILP6 and how information gained from this antibody-antigen interaction may be applicable to drug discovery. The structure determination and binding site mapping showed that binding occurred through the long, structured CDR3 loop and also identified that VHH 67 bound to ILP6 through a novel binding site. This new binding site was confirmed by X-ray crystallography and the functional effects of the VHH 67:ILP6 interaction were ascertained. The relationship between binding affinity and the dynamics of CDR3 was also investigated.
Date of award2015-06-30
Author affiliationDepartment of Biochemistry
Awarding institutionUniversity of Leicester