Inhalation represents a potentially attractive delivery route for biologics, especially those designed to treat pulmonary diseases such as asthma, cystic fibrosis or lung cancer. Delivery directly to the site of action should increase local concentrations of drug, whilst reducing systemic side effects. However, there is limited knowledge regarding the mechanisms of pulmonary clearance, with gaps in understanding; where molecules are absorbed, the mechanisms involved, regional variability throughout the lung, and how to control pulmonary retention and/or facilitate cellular uptake. The work presented in this thesis details the development of a SPECT/CT imaging protocol to determine the pulmonary retention and tissue redistribution of technetium labelled antibodies and their fragments in vivo, in mice, to begin to address these knowledge gaps. The SPECT/CT imaging method was applied to a whole monoclonal murine immunoglobulin G1 (mlgG1), as well as its Fab and scFv fragments and a small protein (FN3) in order to determine whether diffusion controlled pathways were important in pulmonary antibody clearance. Additionally, the pulmonary retention of mutant mlgG1 with differing binding affinities to the murine neonatal Fc receptor (mFcRn) were assessed in order to determine whether antibody transport across the epithelium occurred via active transcytosis. It was determined that 54.4 ± 0.63 % of the total instilled dose of a whole monoclonal antibody remains in the lung over 24 hrs, with Fab and scFv fragments cleared significantly quicker with 28.7 ± 0.73 % and 34.9 ± 0.85 % respectively of the total instilled dose remaining in the lung at 24hrs. The pulmonary retention of the 11 kDa FN3 protein was also assessed with 21.0 ± 0.65 % remaining in the lungs after 24 hrs. No evidence of build up of any protein was detected in the oesophagus/stomach, suggesting little contribution by mucociliary clearance. Very little build up of whole antibody, Fab or scFv was observed in the liver or kidneys. However, very clear evidence of renal filtration of the 11 kDa fragment was observed. There was no difference in the pulmonary retention of wild type IgG and any of the mFcRn binding mutants. Additional investigation of antibody retention rates in the murine house dustmite (HOM) model of asthma, although not making use of the SPECT/CT method, showed that antibody is cleared more rapidly from the diseased lung than the normal lung. It was also shown that the expression pattern of the mFcRn receptor is the same in the normal and HDM exposed lung and so this increase in clearance rate occurs via passive diffusion controlled processes. This is most likely a result of increased paracellular transport due to disrupted mucosal barrier function. The SPECT/CT imaging method developed has proven to be a simple and reliable method to assess, non-invasively, pulmonary antibody retention in vivo. Overall it appears that antibody transport across the pulmonary epithelium occurs predominantly via diffusion controlled mechanisms, which include both paracellular transport and nonspecific transcytosis by pinocytotic routes. Additionally, in the mouse, neither receptor mediated transport by mFcRn nor mucocillary clearance is important in the pulmonary clearance of antibodies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:594271 |
| Date | January 2013 |
| Creators | Pereira, Catherine |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/30444/ |
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