Structural Characterisation of Antibody/Antigen Interactions: Implications for B-Cell Receptor Signalling
thesisposted on 10.02.2017, 13:45 by Philip William Addis
Therapeutic antibodies are an important class of pharmaceutical molecules contributing a significant portion of the total therapeutic market. Due to their high specificity, modular nature and low toxicity, these proteins provide great potential for the development of novel therapeutics for a wide range of diseases. The continued development of improved and unique therapeutics will benefit from improvements in our understanding of the characteristics of these proteins and their functions, facilitating a more informed approach towards therapeutic research and potentially highlighting new molecular information on poorly understood areas of antibody function, such as the initiation of BCR signalling. NMR provides a useful and highly sensitive tool for the investigation of the structural properties of proteins and the changes which occur upon binding. Smaller antibody fragments also provide a useful model for the study of antibody/antigen interactions in solution. The work described in this thesis describes the development of the scFv format of antibody fragment as an amenable and reliable model system from which high quality NMR data can be collected to investigate both the behaviour of the antigen recognising CDR loops and the movements occurring at the interface between the variable domains. An efficient, high yield protein expression method was adapted and developed for the expression of large quantities of 15N/13C/2H labelled scFv which allowed, for the first time, sequence specific backbone assignments for the majority of the scFv in both the free and bound states and the comparison of chemical shift data. Further to this, based upon the domain movements observed, potential mechanisms for the initiation of BCR signalling were investigated resulting in data suggesting the first antigen modulated molecular basis for the coupling of binding from a structurally diverse antigen repertoire to a conserved signalling response.