Kinetics of Inhaled Antibodies by Gamma Scintigraphy

Catherine Pereira1, David Pritchard1, Simon Young2, Carl Webster3. 1University of Nottingham, School of Pharmacy, Boots Science Building, University Park, Nottingham, NG7 2RD, UK, 2AstraZeneca, AstraZeneca Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK, 3MedImmune Ltd., Milstein Building, Granta Park, Cambridge, CB21 6GH, UK.

Inhalation represents a potentially attractive delivery route for therapeutic antibodies, especially those to pulmonary targets, where delivery direct to the local site of action should increase local concentrations of drug, whilst reducing systemic side effects. However, there is limited knowledge regarding the mechanisms of pulmonary antibody clearance, with gaps in understanding: where molecules are absorbed, the mechanisms involved, regional variability throughout the lung, and how best to control pulmonary retention and/or facilitate cellular uptake.

To begin to address these knowledge gaps, we have developed a system using delivery of technetium labelled antibodies and their fragments in vivo (including the 11 kDa FN3 protein, which mimics an immunoglobulin fold and provides data for a molecule smaller than an scFv). The system enables the determination of pulmonary retention and tissue redistribution of inhaled antibodies.

Female balb/c mice (n = 6 per timepoint, weight range 18.2 ± 1.7 g) were dosed by intratracheal instillation with technetium labelled antibody/fragments at 5mg/kg, under isofluorane anaesthesia. Animals were then imaged on a Bioscan nanoSPECT/CT small animal imager, under surgical anaesthesia with 25mg/kg,1mg/kg Ketamine,Medetomidine, whilst spontaneously breathing at 0, 2, 4 and 24 hours post dose. Images were analysed in AMIRA 5.1 and allowed determination of pulmonary antibody retention and tissue redistribution over 24 hrs.

Results show 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. Additionally, 21.0 ± 0.65 % of the FN3 fragment remains in the lungs after 24 hrs. No evidence of build up of any protein was detected in the oesophagus/stomach, suggesting little contribution by mucocillary clearance. Very little build up 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.

The SPECT/CT imaging method developed has proven to be a simple and reliable method to non-invasively assess pulmonary antibody retention in vivo. Work is ongoing to determine the relative importance of paracelluar diffusion and receptor mediated transcytosis by the neonatal Fc receptor (FcRn), as mechanisms by which antibodies are cleared from the lungs.