Production and characterisation of dog CSFl-Fc protein

Abstract

Colony stimulating factor 1 (CSFI) is responsible for maintenance, proliferation and differentiation of cells of the mononuclear phagocyte system, which includes monocytes, macrophages and their progenitor cells. Macrophages are present in all organs and play a role in immunity, organogenesis and tissue remodelling. Macrophages are also the largest extra­hepatic source of insulin-like growth hormone, so indirectly control somatic growth. By controlling differentiation of macrophages, CSFl controls homeostasis. CSFl knockout mice are small, osteopetrotic and have many internal defects. It has been proposed that CSF 1 has therapeutic potential because of the many functions of mononuclear phagocytes. CSFl could be used to treat various indications, including liver failure, bone healing, heart infarction and cancer. However, CSFI has pleiotropic effects and therefore treatment can have many side effects that need to be investigated further. An Fe tag was added to dog CSFl in order to increase its half-life in the circulation, which is essential for therapeutic purposes. The aim of this project is to produce dog CSFl-Fc by expression in mammalian tissue culture cells and assess its biological activity and cross-species reactivity, a first step in developing dog CSFl for testing as a therapeutic for liver disease. Mammalian expression vectors were designed and constructed, then transfected into human embryonic kidney cells. The expression of dog CSFI-Fc was confirmed by western blots and mass spectrometry. Dog CSFI-Fc was produced by large-scale cell transfections and purified by affinity chromatography followed by size exclusion chromatography. Biological activity assays showed that purified dog CSFI­Fc was biologically active and cross-reacted with non-canine CSFI receptors, which also confirmed that the protein is highly functionally conserved. The ultimate goal of this project is to develop dog CSFI-Fc as a biological therapeutic for dog liver failure and further tests in vivo are required.