Chemical and Biomolecular Engineering - Theses
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ItemNanoengineering Antibody Assemblies for Biomedical Applications( 2021)The clinical success of monoclonal antibody therapy has inspired research in understanding the fundamental molecular basis of antibody-antigen interactions and the engineering of antibodies with enhanced or novel properties. With the emergence of nanomedicine, antibodies have been widely applied as targeting ligands decorated on the surface of therapeutic nanostructured modalities – including liposomes, protein nanoparticles, and polymeric assemblies – for drug delivery and imaging applications. However, little is known about how antibodies assembled in a cluster or particulate form interact with antigens in a biological system, largely due to the challenge in preparing ‘pure’ antibody assemblies with controlled physicochemical properties. In this thesis, a mesoporous silica template-mediated assembly platform was applied to fabricate well-defined nano-assemblies of therapeutic antibodies, including conventional monoclonal antibodies and antibody-drug conjugates. The antibody nano-assemblies (AbNAs), crosslinked with poly(ethylene glycol)-N-hydroxysuccinimide (PEG-NHS), preserved the selectivity of the monoclonal antibody and induced receptor-mediated internalization of antibodies to achieve enhanced intracellular response, such as growth inhibition. This strategy presents opportunities for intracellular delivery of monoclonal antibodies, as well as a versatile platform for fundamental studies on the interactions between antibody assemblies and cells. Facile engineering of AbNAs can be achieved by leveraging the intrinsic property of the PEG crosslinker, such as chain architecture (PEG arm numbers and arm length), to regulate bio-nano interactions. As a widely recognized stealth material, PEG can prolong blood circulation time to allow the accumulation of nanoparticles in target tissues, however, it could also result in decreased targeting efficacy by blocking the antigen-binding sites. This thesis investigates the influence of PEG crosslinking, specifically the effect of using PEG crosslinkers with different chain architecture on the formation of AbNAs and their bio-interaction with respect to specific binding and uptake by phagocytic cells. PEG crosslinkers with less arms but longer arm length were found to be more beneficial for AbNAs to achieve both minimal phagocytic capture and optimal targeting. Furthermore, the targeting efficacy of AbNAs could be enhanced by substituting conventional monoclonal antibodies with engineered antibody fragments. Nanobodies, also known as single-domain antibodies (sdAb), are the smallest antigen-binding unit (12-15 kDa) that solely bind to the target antigen. The unique structure of nanobodies offers several desirable features, including small size, high stability, strong antigen-binding affinity and low immunogenicity, which makes nanobodies superior for antibody nano-assembly engineering. The nanobody nano-assemblies (NanoNAs), prepared via the template-mediated assembly platform, exhibited significantly enhanced selective association to target cells and reduced phagocytic association in comparison with full-sized AbNAs, owing to the unique structure of nanobodies that allowed a large amount of active binding sites to be presented on the particle surfaces and eliminated crystallisable fragment (Fc) receptor-mediated capture by phagocytic cells. Overall, the versatile antibody nano-assembly systems expanded our understanding of antibody-antigen interactions, and provides a facile platform to engineer antibody assemblies with novel or enhanced properties for biomedical applications.