Structure-based Characterization And Preclinical Pharmacolgy Evaluation Of PEGylated Canine-human Chimeric Uricase

Uricase [EC 1.7.3.3] is an enzyme involved in the purine degradation pathway and catalyzes the hydrolysis of uric acid to the more water-soluble allantoin. Human beings lack active uricase because of gene mutations, leading to uric acid as the end product of purine metabolism and resulting in the development of urate nephropathy and gouty arthritis when the concentration of uric acid reaches its solubility limit in blood (hyperuricemia). Both the natural (UricozymeTM) and recombinant (ElitekTM) Aspergillus Flavus uricases had been used therapeutically in patients with acute hyperuricemia but their high immunogenicity and short half-life has limited their long-term use for treating chronic hyperuricemia and associated diseases (gout). PEGylated uricase, with low immunogenicity and a long circulation half-life has been under preclinical investigation since 1981, but there has been only one commercial PEGylated uricase treatment (KrystexxaTM) used in clinical therapy. However,92% of patients developed antibodies and 57% of patients showed decreased urate-lowing efficacy after repeated administrations in the clinical trials of Krystexxa. Moreover, the only commercially marketed 10 kDa mPEG modified porcine-like uricase can only be used for intravenous infusion. As far as we know, to date no form of uricase or PEGylated uricase has been developed that has sufficiently reduced immunogenicity for safe and reliable use in chronic therapy.In this thesis, we first screened and finalized canine-human chimeric uricase with high enzymatic activity, recovery and stability than the original wild-type canine uricase. Homogeneous recombinant tetrameric chimeric uricase was successfully purified and then modified to saturation with a tailored 5 kDa mPEG-SPA. The preclinical pharmacology and safety properties of the PEGylated canine-human chimeric uricase was investigated in vivo.Section one:Construction, screening and structure-based characterization of canine-human chimeric uricaseCanine uricase was first selected for recombinant expression and production in E.coli, due to its high catalytic activity and identity with deduced human uricase. Five chimeric uricase uricases based on the wild-type canine uricase were constructed and characterized. Both the activity and stability of the finalized uricases were improved compared to the original uricase. Homology modeling was used to analyze the enhancements:the C-terminus of mammalian uricase (residues 291-304) is important for maintaining the activity and stability of the protein, whereas the lower domain of H4 and the upper part of S7 (residues 245-253) are associated with the recovery and stability of tetrameric uricase. In addition, substrate docking and molecular dynamics simulations were performed to confirm the active center of the chimeric uricase uricase. A novel oxygen binding architecture was clarified and the function of several highly conserved residues was investigated for the first time. The amino acid sequence was confirmed by using peptide mapping. A novel active non-disulfide covalent cross-linked dimer, which was widely existed in uricase family, was first structurally confirmed by using peptide mapping, size exclusion HPLC and affinity chromatography. The statues and function of the highly conserved cysteine residues in mammalian uricase were investigated by blocking free cysteine, then digested with trypsin and analyzed using HPLC-MS methods.Section two:Development of novel PEGylated mammalian uricase with sufficiently reducing of immunogenicityTetrameric and large aggregated uricase proteins were successfully purified and characterized. In the subsequent pegylation process, anion-exchange chromatography was employed to remove the PEG Diol from PEG reagents. Uricase with or without aggregates was modified to saturation with purified or unfractionated 5 kDa mPEG-SPA, respectively, resulting in three kinds of PEGylated uricases. Large uricase aggregates could affect enzymatic retention but the impact could be resolved after removing the aggregates and the enzymatic retention of PEGylated tetrameric uricase was higher than 85%. Dynamic light scattering and transmission electron microscope were first employed to analyze the size distribution of different PEGylated proteins. The impact of unmodified uricase aggregates and cross-linked uricase congregates induced by PEG Diol on pharmacokinetics and immunogenicity were studied in vivo. The accelerated blood clearance phenomenon previously identified in PEGylated liposomes, also appeared in rats injected with PEGylated uricase aggregates. Anti-PEG IgM antibodies rather than neutralizing antibodies were found to mediate the accelerated blood clearance. Moreover, we confirmed for the first time that the size of conjugates is of primary important in triggering such phenomena. After removal of uricase aggregates and PEG Diol, accelerated blood clearance could be successfully avoided. Section Three:Preclinical pharmacology and safety evaluation of 5 kDa mPEG modified canine-human chimeric uricaseThree different animal models were first set up to evaluate the urate-lowering efficacy, preventive effect on urate nephropathy and therapeutic effect on gouty arthritis, respectively. The pharmacokinetic properties and the bioavailability of subcutaneous injection of mPEG-UHC were evaluated in rodents and non-human privates. The elimination half-life of intravenous injection of mPEG-UHC in rats and monkeys were 34.9h and 134.3h. The systemic bioavailability of mPEG-UHC after subcutaneous administration in the above two species is higher than 60%, indicating that subcutaneous injection may be regarded as a candidate administration route. According to 28-day toxicity studies, there were no test article-related effects on clinical observations, hematology and blood biochemistry test. The only treatment-related histopathological changes were the vacuolation of splenic macrophages in one middle-dose (12.5%) and two high-dose (25%) rats, which may be caused by the inducing of anti-PEG IgM antibody and triggering of the uptake of mPEG-UHC by the mononuclear phagocyte system in spleen, but such change did not bring any pathology changes and functional lesions. This findings not only provide a worthy drug candidate for treating gout, but also represent a valuable resource for understanding the structure and catalytic mechanism of the whole uricase family, the mechanism of accelerated blood clearance triggered by PEGylated proteins and nanoparticles, the elimination mechanism and safety of large PEGylated proteins.