McDonnell, TCR;
(2016)
Does PEGylated DI have the potential to be an effective therapeutic for Antiphospholipid Syndrome?
Doctoral thesis , UCL (University College London).
Abstract
Antiphospholipid Syndrome (APS) has 0.3-1% population prevalence and characterised by clinical phenotypes (thrombotic and obstetric) and the presence of anti-phospholipid antibodies (aPL). APS is the leading cause of strokes in patients <50 years old and causes recurrent miscarriage. Current treatment is inadequate and consists of life long non-specific anticoagulant therapy. It has been previously proven that Domain I (DI) of Beta-2-glycoprotein I (β2GPI) harbours the dominant epitope for pathogenic antibodies in APS and administration of a recombinant version of DI can abrogate clotting In an APS mouse model. Despite this, DI requires chemical modification to be suitable for drug administration. This thesis details the production methodology, characterisation and results of chemical conjugation of DI to poly ethylene glycol (PEG). Production remains a difficult hurdle in the production of biological based therapeutics. I increased production to 75 mg/L (from 4-6 mg/L). I then semi-automated the folding process, increasing yields and generated purification protocols yielding a purity of >95% throughout. PEGylation was undertaken to extend half life and reduce immunogenicity. I achieved conjugation yields of ~ 50% mono-PEGylated species (molar yield) with minimal waste Di-PEGylated product. Expression tag was removed and endotoxin removed successfully and prepared for final testing. Activity testing included a novel thrombotic functional test, two competition ELISAs (IgG (n=6) & IgA (n=4) ) and a mouse model. All assays showed a high level of retained activity for the protein. Using a novel functional, thrombotic test (n=4), results suggested a gain of function for WT PEGylated species but not PEGylated mutant DI species. In the IgG ELISAs on average >80% activity was retained with similar seen in the IgA assays. Together, these results suggest that PEGylated DI harbours the potential to be the first specific, targeted therapeutic in APS.
Archive Staff Only
 |
View Item |
