AbstractNanoparticles (NPs) have conferred some benefit in improving the outlook in patients diagnosed with pancreatic adenocarcinoma (PDAC) with the encapsulation of chemotherapeutics such as paclitaxel and irinotecan, minimising systemic toxicities. NP success in the treatment of PDAC could hypothetically be enhanced through the attachment of a ligand. Though no actively targeted NP has been approved in the clinic, previous endeavours have identified the importance of site-specific conjugation to optimise paratope display. In addition, the advantages of antibody-fragments in place of full monoclonal antibodies (mAbs), in limiting steric hindrance on the NP scaffold and negating Fc-mediated immunogenicity has been reported. Though still in their clinical infancy, shark derived antibody-like fragments, variable new antigen receptors (vNARs), have displayed numerous characteristics making them an ideal targeting moiety for NPs in cancer therapy. These attributes include their small size (12 kDa), high affinity for their cognate antigen, and amenability to reformatting to allow for site-specific conjugation to the NP scaffold.
The first body of work described in this thesis explored the optimisation of the single emulsion formulation to enhance loading of doxorubicin (DOX) within polymeric NPs, with the produced formulation possessing ideal physicochemical characteristics for drug delivery, with the particles capable of a ‘smart’ DOX release and induced cell death in pancreatic cancer cell lines as determined by CellTitre® Glo.
Secondly, a range of potential targets for PDAC therapy were identified using publicly available datasets with a lead target selected. Both solid and soluble phage display panning was performed to identify potential binders against the selected lead target using Elasmogen’s proprietary semi-synthetic vNAR libraries however majority of selected clones possessed frameshift mutations or stop codons within their antigen binding domains.
Thirdly, the site-specific conjugation of vNARs to maleimide functionalised NPs was assessed with the vNAR clones reformatted to include a single free cysteine and allow for the formation of a thioether bond. The reformatting resulted in the loss of target binding as determined via FLISAs and consequently, homodimer constructs of these binders were engineered with a free cysteine located within the linker, displaying target specific binding as determined via ELISA. However, this binding was lost following its conjugation to maleimide-NPs, with potential issues in linker design. Finally, clone 3E01, was isolated from Elasmogen’s vNAR libraries against a novel target that was identified from the publicly available datasets and engineered to include a free cysteine on its C-terminus (C-3E01), retaining binding. This C-3E01 clone was successfully conjugated to the DOX-NPs described in Chapter 3 (with maleimide functionalisation) showing target specific binding and enhanced the anti-tumour activity of the DOX-NPs in target expressing pancreatic cell lines as determined via CellTitre® Glo and clonogenic assays, indicating a future for vNAR-targeted nanosystems in the treatment of PDAC.
To summarise, the work presented within this thesis, describes the isolation and engineering of vNARs for the development of actively targeted NPs and the potential of these nanosystems as the next-generation of therapeutics for pancreatic cancer therapy.
Thesis is embargoed until 31 July 2028.
|Date of Award||Jul 2023|
|Sponsors||Northern Ireland Department for the Economy & Elasmogen Ltd|
|Supervisor||Daniel Longley (Supervisor) & Christopher Scott (Supervisor)|