Nogueira, João Carlos Faria; (2020) Forming Next-Generation Antibody-Nanoparticle Conjugates through the Oriented Installation of Antibody Fragments. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Use of antibody-nanoparticle conjugates (ANCs) has emerged as a multi-disciplinary strategy for combating cancer - they combine the versatility of nanoparticles and the potential to deliver cargo to cancer cells with the high targeting specificity of surface antibodies to recognise specific biomarkers that are expressed in cancer cells. Several strategies have been employed to graft nanoparticles to antibodies, however, most of them rely on fragile non-covalent interactions or on methods that do not exert control on antibody paratope orientation (e.g. random modification of multiple lysine residues on antibodies). These issues greatly limit ANCs antigen binding capability, reproducibility and, thus, overall efficacy. In this thesis, alternatives strategies of generating ANCs are proposed, regarding antibody orientation on the nanoparticles’ surface through the use of pyridazinedione-based linkers that site-selectively modify disulfide(s) on antibodies. The overall aim is to achieve highly-controlled ANC construction so that these next-generation ANCs can be employed in future cancer treatments. In Chapter 1, an introduction to current protein modification techniques is presented and, in a more biological context, the structure and use of full antibodies and antibody fragments is described. Additionally, an overview of the current biomedical applications of numerous different types of inorganic and organic nanoparticles is introduced. In Chapter 2, the creation of antibody fragment Fab targeted PEG-PLGA nanoparticles is reported. In particular, the generation of Trastuzumab Fab fragments via digestion techniques and a new approach for their attachment to PEG-PLGA nanoparticles and the consequent results of improved antigen binding are described. In Chapters 3 and 4, different proteins are employed for the generation of ANCs, namely Cetuximab Fab (in which cell studies are also performed) and considerably smaller proteins such as variable new antigen receptors (VNARs) via a similar methodology to that employed in Chapter 2. Concluding, an overview of achieved results and future work are covered.

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