Protein-polymer conjugation improves pharmacokinetics and pharmacodynamics and has successfully been used to improve the therapeutic value of proteins. Polyethylene glycol (PEG) has been the considered as the golden standard in protein-polymer conjugation since the pioneering discovery by Abuchowski et.al in 1977. On the other hand, an alarming 42% of healthy humans have anti-PEG antibodies as reported by Yang et. al in 2015. <i>Pegloticase</i> (PEG-uricase) used for the treatment of gout was met with 38% patients eliciting anti-PEG antibodies in Phase I and 89% patients in the Phase III trials leading to a loss of treatment response, increased drug clearance and risk of infusion reactions and adverse events in at least 90% of the patients. This demands the development of an alternative uricase bioconjugate as a potential alternative of PEG-uricase.<br/>To achieve this, we are engineering ROMP derived water soluble polynorbornene (PNB) polymers. Two different PNB based polymers, (i) PEG-based PNB and (ii) Zwitterionic PNB (Polyzwit- PNB) were synthesized and covalently conjugated to the lysine residues of homo tetrameric uricase enzyme. Pegylated (10kDa)-uricase was also synthesized by graft-to conjugation technique as a control to compare our new PNB-uricase conjugates to the state-of-the-art enzyme therapy for gout treatment. The successful conjugation was confirmed by SDS-PAGE and a lower retention volume on the FPLC compared to the unmodified uricase. The purified uricase conjugates were analyzed by enzyme activity assay and compared to the unmodified uricase enzyme. The immune recognition studies of the conjugates were done to study the binding ability of uricase antibody to the uricase conjugates. We expect that our PNB-uricase conjugates will have a reduced antibody recognition compared to the unmodified uricase enzyme. Additionally, it eliminates the chances of polymer specific anti-PEG antibody binding.

Engineering graft-to polynorbornene based uricase conjugates with low immunogenicity

ACS Spring 2022

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