Rationale: Over the last 20 years, granulocyte colony-stimulating factor (GCSF) has become a recognised therapy in the treatment of patients suffering from neutropenia. Current therapies require daily injections of GCSF to stimulate stem cell production and response to treatment is often unpredictable as GCSF is rapidly cleared. A number of approaches to reducing GCSF clearance have been tried mainly through conjugation with another moiety. The technologies already being employed, included PEGylation, immunoglobulins and glycosylation to increase the half-life of rhGCSF, however although these approaches have reduced clearance the pharmacokinetic profile has remained unpredictable. The aim of this project is to create a long acting GCSF with predictable pharmacokinetic profile to provide a more effective treatment for generating stem cells for bone marrow transplantation.
Hypothesis: The incorporation of variable glycosylated linkers between two GCSF molecules will create a construct with high molecular weight and protected from proteolysis resulting in reduced clearance while retaining bioactivity.
Methodology: GCSF tandem molecules with linkers containing between 2 and 8 NAT glycosylation motifs and their respective controls (Q replaces N in the sequence motif NAT) were cloned, and sequenced. Following expression in Chinese hamster ovary (CHO) cells, expressed protein was quantified by ELISA and analysed by SDSPAGE and western blot to confirm molecular weights. In vitro bioactivity was tested using an AML-193 proliferation assay. Immobilised Metal Affinity Chromatography (IMAC) was used to purify the protein. Pharmacokinetic and pharmacodynamic of the GCSF tandem proteins were measured in normal Sprague Dawley Strain rats with full ethical approval.
Results: Purified glycosylated tandem molecules show increased molecular weight above that of controls when analysed by SDSPAGE. All GCSF tandems show increased bioactivity in comparison to rhGCSF. Following intravenous administration to rats, GCSF2NAT, GCSF4NAT and GCSF8NAT containing 2, 4 and 8 glycosylation sites respectively displayed a reduced rate of clearance compared to both rhGCSF and non-glycosylated GCSF tandem control. Both GCSF2NAT and GCSF4NAT show a significant increase in the percentage of neutrophils over controls at 12 h post injection. This effect however is short lived as the counts at 24+ h are not significantly different to controls. GCSF8NAT shows an increase in the percentage of neutrophils that is only significant at 48 h.
Conclusion: Results show that the use of glycosylated linkers to generate GCSF tandems results in molecules with increased molecular weight, improved in vitro bioactivity, longer circulating half-lives and enhanced neutrophilic population when compared to both rhGCSF and the non-glycosylated tandem protein.