A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties

Abstract

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.

Thermostable and Long-Circulating Albumin-Conjugated Arthrobacter globiformis Urate Oxidase

“…Gout is a common type of inflammatory arthritis in adults, resulting from the formation of uric acid crystals in the joints and other tissues [ 1 , 2 , 3 ]. Therefore, the treatment of gout has focused on reducing serum uric acid levels, which has been effectively achieved by the injection of urate oxidase [ 3 , 4 ]. Urate oxidase (Uox, Enzyme Commission number: 1.7.3.3) is a peroxisomal liver enzyme that catalyzes the conversion of insoluble uric acid (0.06 g/L) to the more water-soluble 5-hydroxyisourate (10.6 g/L; predicted using ALOGPS) [ 5 , 6 , 7 , 8 ].…”

“…However, the clinical applications of HSA-conjugated AfUox may be limited by its intrinsic immunogenicity and low thermostability [ 23 ]. Recently, Uox derived from Arthrobacter globiformis (AgUox) was determined to hold desirable properties for therapeutic development, including soluble expression in Escherichia coli , good solubility at neutral pH, low immunogenicity, and good thermostability [ 4 , 24 ]. We confirmed that wild-type AgUox is more thermostable than wild-type AfUox ( Figure S1 ).…”

Molecular Elucidation of a Urate Oxidase from Deinococcus radiodurans for Hyperuricemia and Gout Therapy

“…Moreover, size-exclusion chromatography (SEC) revealed that DrUox is a tetrameric protein in the native state ( Figure 2 B). Thus, DrUox may form a canonical homo-tetramer as do other urate oxidases [ 26 ].…”

“…Urate oxidase activity was assayed by monitoring the depletion of uric acid, which was detected by a decrease in absorbance at 293 nm [ 20 , 26 , 47 ]. The optimal temperature of DrUox was evaluated by incubating 0.5 μM DrUox and 500 μM UA in a temperature range from 10 to 80 °C for 3 min in pH 9.0, then the residual activity was assayed.…”

In vitro models for immunogenicity prediction of therapeutic proteins

“…In in vitro PBMC assays, the antigen priming of CD4+ T cells by mature MoDCs leads to the proliferation of CD4+ T cells. T cell proliferation is followed by radioactive labeling with tritiated thymidine pulsation and scintillation counter [75, 77,85], or by 425 using more sensitive labeling with fluorochromes like carboxyfluorescein succinimidyl ester (CFSE) and 5-ethynyl-2'-deoxyuridine (EdU) in combination with flow cytometry [86,87].…”