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Cusin, Lola M. L. (2025) Development Of a nanobody-conjugate platform for targeted drug delivery in airway hypersecretory diseases. PhD thesis, University of Nottingham.

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Abstract

Airway Hypersecretory Diseases (AHDs) are conditions in which the main pathophysiology is mucus hypersecretion. The incidence of AHDs is high (over 500 million affected worldwide) and comes with a diminished quality of life for patients. Very few treatment protocols focus on mucus hypersecretion. We propose a nanobody-conjugate platform for the targeted treatment of AHDs based on nanobody targeting of goblet cell surface antigens and a site-directed conjugation method to allow therapeutics to be attached to the nanobodies.

A diverse synthetic nanobody library based on the shark vNAR 5A7 scaffold and expressed as N-terminal pIII fusion proteins within the phagemid pSEX81 was developed to create a naïve nanobody library displayed on M13-based bacteriophage particles, for screening. NGS analysis confirmed a diversity of 2.56 x 109, comparable to libraries described in the literature.

Human bronchial epithelial cells (HBEpc) were cultured at the air- liquid interface as a model for the human airway epithelium, and IL-13 stimulation was used to simulate the hypersecretory phenotype observed in AHDs. Flow cytometry analysis validated IL-13 driven airway remodelling, and goblet cells metaplasia. Increases in the percentage of goblet cells in culture were observed, similar to the effects reported in the literature (from 3.77% to 17.92% post IL-13 stimulation, N=3). A whole cell biopanning protocol was designed for phage display selection of potential binders to stimulated epithelial cells and yielded 8 clones. Sequencing of these clones revealed highly disturbed origin of replication and nanobody regions, and no binders could be identified.

Functionalisation of nanobodies by site-specific bioconjugation of a fluorescent tag was investigated. Sortase A catalysed coupling was selected as it allowed for the conjugation of the fluorescent label selectively at the C-terminus, preventing the labelling of the binding sites of nanobodies. A Cy5.5 label was synthesised by solid-phase peptide synthesis and amide coupling. The label encoded the oligoglycine recognition site of Sortase A enzyme, and the cyanine fluorophore was coupled to the side chain of a lysine. The original vNAR 5A7 was engineered to express the LPXTG recognition sequence of Sortase A alongside a poly-histidine tag for purification by immobilized metal affinity chromatography. Despite several optimisations of the expression protocol, the Hen egg white lysozyme specific vNAR 5A7 was not expressed in E. coli, and further work will be needed to produce the protein. The challenges of whole cell biopanning and nanobody expression and purification are discussed.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Tighe, Patrick J. Mitchell, Nicholas
Keywords:	Airway hypersecretory diseases (AHDs), Nanobody-conjugate platform, Drug delivery
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QT Physiology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	81685
Depositing User:	Cusin, Lola
Date Deposited:	10 Dec 2025 04:40
Last Modified:	10 Dec 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/81685

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