The colony-stimulating factor-1 receptor (CSF-1R) is expressed by the mononuclear phagocytic lineage, and is important for the development of these cells from their progenitors and also for promoting their survival and activation after maturation. The receptor has two ligands, CSF-1 and IL-34, which induce the formation of a stable dimer between two receptor monomers. This leads to intracellular autophosphorylation of tyrosine residues and subsequent signalling cascades, leading to rapid protein expression, cytoskeleton remodelling and cellular motility. Although CSF-1R signalling is crucial for normal embryogenic development and other physiological functions mediated by the phagocytic lineage, it has also been found to promote the pathogenic progression of cancer. Tumour-associated macrophages (TAMs) can comprise a large proportion of the cellular population in several solid tumours. These cells promote several hallmarks of cancer malignancy, such as increased neovascularization, tissue invasion, induction of metastases and immunosuppression. In this work, it was confirmed that CSF-1 had a prominent role in inducing cancer-promoting cellular phenotypes. Both canine cancer cells and macrophages respond to this cytokine, respectively increasing cancer cell proliferation and reducing inflammatory activation. Given the importance of CSF-1R signalling in the tumour microenvironment, antibodies were generated with the objective of blocking receptor function. Mice were immunized with either the extracellular region or the dimerization domain of the CSF-1R. Hybridomas were produced using the primed splenocytes, and monoclonal antibody (mAb) candidates were selected based on their performance in immunostaining and on their capacity to inhibit CSF-1R+ cells. The best antibodies were subjected to speciation. Chimeric antibodies maintained the ability of the parental mAbs to inhibit macrophage proliferation following CSF-1R stimulation. However, the mAbs possessed moderate affinity and specificity for their target, failing to stain monocytes and presenting a degree of cross-reactivity. The binding properties of one of such mAbs were altered by PCR-induced mutations, generating semi-synthetic antibody libraries. These were screened by phage display, yielding novel clones that show reduced cross-reactivity with unrelated proteins and retain the property of inhibiting macrophage survival. These results are a step in the development of therapeutic monoclonal antibodies for cancer treatment in dogs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:702239 |
| Date | January 2015 |
| Creators | Beirão, Breno Castello Branco |
| Contributors | Argyle, David ; Hume, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/19529 |
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