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Generation of long acting therapies using glycosylated linkers

Abdulrahman Alshehri R J Ross and I R Wilkinson University of Sheffield, UK

Introduction: Rationale: The current therapeutic drugs such as, growth hormone (GH), granulocyte colony
stimulating factor (GCSF) and leptin require once daily injections, which are inconvenient and expensive. Therefore, a
number of approaches to reducing therapeutic regimens clearance have been tried mainly through conjugation with
another moiety. One such technology already being employed is PEGylation; however this has been shown to be nonbiodegradable
and toxic. A previous study by Asterion has shown that the use of glycosylated linkers between two GH
ligands to create protein tandems resulted in their glycosylation and an increased molecular weight (MW) whilst
maintaining biological activity. The use of this technology using GCSF as an example will be presented, but can be
easily applied to other molecules such as leptin. Hypothesis: The incorporation of variable glycosylated linkers
between two GCSF ligands will create a construct with high molecular weight and protected from proteolysis resulting
in reduced clearance without blocking bioactivity.
Methodology: GCSF tandems with linkers containing between 2�8 NAT glycosylation motifs and their respective
controls (Q replaces N in the sequence motif NAT so there is no glycosylation) were cloned, and sequenced. Following
expression in Chinese hamster ovary (CHO) cells, expressed protein was analysed by SDS PAGE to confirm
molecular weights. In vitro bioactivity was tested using an AML�193 proliferation assay. Immobilized metal affinity
chromatography (IMAC) was used to purify the protein. Pharmacokinetic and pharmacodynamics properties of the
purified GCSF tandem proteins were measured in normal Sprague Dawley rats with full ethical approval.
Results: Purified glycosylated tandems show increased molecular weight above that of controls when analysed by
SDS PAGE. All GCSF tandems show increased bioactivity in comparison to native GCSF. Following intravenous
administration to rats, GCSF2NAT, GCSF4NAT, GCSF8NAT containing 2, 4 & 8 glycosylation sites respectively and
GCSF8QAT (non-glycosylated GCSF tandem control) showed approximately 3�fold longer circulating half�life
compared to that reported for the native GCSF (1.79 hours). Both GCSF2NAT and GCSF4NAT show a significant
increase in the percentage of neutrophils over controls at 12 hours post injection. This effect however is short lived as
the counts at 24+ hours are not significantly different to controls. GCSF8NAT shows an increase in the percentage of
neutrophils that is only significant at 48 hours. 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 native GCSF and the nonglycosylated
tandem protein