Improved Pharmacokinetic and Biodistribution Properties of the Selective Urokinase Inhibitor PAI-2 (SerpinB2) by Site-Specific PEGylation: Implications for Drug Delivery

Purpose Overexpression of the serine protease urokinase (uPA) is recognised as an important biomarker of metastatic disease and a druggable anticancer target. Plasminogen activator inhibitor type-2 (PAI-2/SerpinB2) is a specific uPA inhibitor with proven potential for use in targeted therapy. However, PAI-2 is rapidly cleared via the renal system which impairs tumor uptake and efficacy. Here we aimed to improve the pharmacological properties of PAI-2 by site-specific PEGylation. Methods Several cysteine to serine substitution mutants were generated for PEGylation with PEG-maleimide (size range 12–30 kDa) and the physico-chemical and biochemical properties of the PEG-PAI-2 conjugates characterised. Radiolabeled proteins were used for evaluation of blood clearance and tissue uptake profiles in an orthotopic breast tumor xenograft mouse model. Results PEGylation of the PAI-2 C161S mutant gave a predominant mono-PEGylated-PAI-2 product (~90%) with full uPA inhibitory activity, despite a significant increase in hydrodynamic radius. Compared to un-PEGylated protein the plasma half-life and AUC for PEG 20 -PAI-2 C161S were significantly increased. This translated to a 10-fold increase in tumor retention after 24 h compared to PAI-2 C161S , an effect not seen in non-target organs. Conclusions Our data underscores the potential for PEG 20 -PAI-2 C161S drug conjugates to be further developed as anti-uPA targeted therapeutics with enhanced tumor retention.