Assessment of a strategy to inhibit clearance of proteins by alveolar macrophages
(2009)
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- Authors
- Supervisors
- Vanbever, Rita
- Abstract
- Inhalation aerosols are proving to be a promising alternative to injection for the systemic administration of peptides and proteins. The lungs provide higher bioavailabilities of macromolecules than any other non-inasive routes of delivery. However, bioavailabilities of inhaled therapeutics remain limited and do not exceed 10 % in general, suggesting that uncontrolled biological losses in the respiratory tissue significantly diminish molecular transfer to the bloodstream. Recently, alveolar macrophages (AM) have been shown to be a primary barrier to pulmonary absorption of macromolecules. The uptake and the degradation of inhaled macromolecules by AM competes with their systemic absorption, and thereby, lowers bioavailability to the degree that the rate of degradation is near to or greater than the rate of systemic absorption. The aim of this thesis was to assess an AM-inhibiting strategy in order to decrease AM uptake and clearance of proteins and thereby, increase their pulmonary absorption to the systemic circulation. We aimed to create a sterical hindrance around AM to repel the approach, the binding and the endocytosis of therapeutic proteins. The sterical hindrance was created by grafting poly(ethylene glycol) (PEG) chains on the cell surface of AM using PEGylated monoclonal antibodies (mAbs) directed to sialoadhesin (Sn), a macrophage-restricted adhesion molecule. Anti-mouse Sn mAbs, SER-4 and 3D6, were conjugated to PEG 5 kDa and PEG 20 kDa, resulting in the incorporation of up to 3 molecules of PEG per mAb molecule. PEGylation of antibodies had little effect on their antigen binding activity. Yet, PEGylation strongly increased the potency of antibodies to inhibit red blood cells adhesion mediated by Sn. We concluded that the increase of the potential of SER-4 and 3D6 antibodies by PEGylation was likely due to the steric hindrance provided by the PEG molecules. Sn expression on mouse AM, the population of macrophages targeted by the strategy, was then evaluated. Although Sn expression is restricted to macrophages, it is limited to specific subsets of tissue macrophages and no data clearly demonstrate Sn expression on mouse AM. We showed using flow cytometry that Sn was expressed on murine AM and that this receptor was regulated by cis-interactions. Finally, in order to assess the macrophage-inhibiting strategy in vitro, we used thioglycollate-elicited peritoneal macrophages (TPM) induced to express Sn. These cells can be obtained in large quantities and they show a rapid induction of Sn expression. We first characterised endocytosis modes of albumin and transferrin in these cells and we showed that these molecules were taken up by non-specific adsorptive endocytosis and specific receptor-mediated endocytosis, respectively. Latex beads were also confirmed to be internalised by phagocytosis in TPM induced to express Sn. We showed that PEGylation of macrophages surface using PEGylated SER-4 and 3D6 mAbs did not impair membrane integrity nor cell metabolism and that this local Sn-mediated PEGylation and the associated steric effects of PEG inhibited neither pinocytosis of macromolecules nor phagocytosis of particles. In conclusion, this thesis work showed that PEGylation of Sn on macrophage surface using PEGylated anti-Sn mAbs was not effective to repel the approach, the binding and the uptake of macromolecules and particles. PEGylation of the cell surface needs to be improved in order to make possible our approach of AM inhibition. Yet, our results have however shown for the first time, that PEGylation of antibodies greatly increases their efficacy to inhibit cell adhesion activity. Flexibility and motility of PEG molecules on SER-4 and 3D6 mAbs most likely created a steric hindrance around antibodies that increased the inhibition of red blood cells specific binding to Sn. Thus, PEGylation of antibodies directed to cell surface receptors could be potentially exploited in a therapeutic setting, such as autoimmune or inflammatory diseases, to increase the inhibitory potency of antibodies without impairing vital functions of cells.
- Affiliations
Citations
Ducreux, J. (2009). Assessment of a strategy to inhibit clearance of proteins by alveolar macrophages. https://hdl.handle.net/2078.5/112037