AbstractConsiderable success in the use of monoclonal antibodies (mAbs) as a targeted approach to cancer therapy has stimulated rapid development in this field of research. Due to the complex nature of the tumour microenvironment many novel antibody formats have been designed to expand on the therapeutic capacity of immunoglobulin G (IgG).
One important example is the development of antibody-nanoparticle conjugates. Here, the antibody structure was exploited by producing antibody-based ferritin fusion proteins. The protein cage architecture of the iron storage protein ferritin was utilised by fusing it to antibody variable domains to create a targeted biological nanoparticle. It was hypothesised that on generation of these novel constructs, it would be possible to demonstrate their potential for use in cancer diagnostics and/or therapy. Thus, the aims of this thesis were to produce and characterise antibody-ferritin fusion proteins with specificity towards non-biological hapten NIP (4-hydroxy-3-iodo-5-nitrophenylacetate) and for CD33, an established target for certain leukaemias. Structural and functional characterisation of these proteins would act as an initial assessment as to the suitability of these constructs in a cancer setting. Production and purification techniques were initially established for both antibody-ferritin fusion proteins followed by characterisation of structural and binding attributes. This analysis revealed that these proteins assemble to form multivalent structures and retain the capacity to bind to immobilised antigen in a similar concentration range to their parent IgG. However, the antibody-ferritin fusion proteins appeared to have the ability to interact with tumour cell lines via ferritin receptors, which will bring additional complexity to their development as diagnostic and/or therapeutic agents.
A second next-generation antibody format is the bispecific antibody (biMab) - an antibody with the ability to bind to two independent epitopes. In this study IgG constructs with single point mutations were engineered to enable the formation of biMabs using DuoBody technology. Here, the central hypothesis was that production of these IgG mutant constructs would enable the formation of biMabs which would subsequently act in bridging immune cells and target cells in cell culture models. The aim was to carry out controlled Fab-arm exchange to generate bispecific antibodies with specificity towards the immune cell receptor CD64 and NIP. Successful DuoBody formation revealed the capacity of these biMabs to bring CD64-expressing immune cells and target cells (in this case derivatised with the NIP antigen) into close proximity for immune-mediated cell destruction. Further, a similar construct lacking the site for N-linked glycosylation at position 297 and therefore with reduced effector function was produced. Functional comparison of these biMabs highlighted a role for the Fc region in enhancing biMab induced immune-mediated target cell destruction.
Overall, the results in this thesis provide a platform for the production of two next-generation antibody-based structures with potential in cancer diagnostics and/or therapy. It was possible to produce and characterise these constructs to address the project hypotheses. However, results highlighting intricacies in the binding characteristics of antibody-ferritin fusion proteins were not foreseen and require further analysis.
|Date of Award||2016|
|Supervisor||Jennifer Woof (Supervisor) & Prabhjyot Dehal (Supervisor)|