Table 1.
Uricase sequence identity comparison.
Fig 1.
Expression of candidate uricase in E. coli. 8 uricase sequences were expressed in E. coliand evaluated for soluble (S) and insoluble (P) expression level.
As shown in Fig 1, most uricases were present at high level in the insoluble (P) material. The pig-baboon chimera appears to express almost entirely in the insoluble (P) fraction (Lane 9). Cytosolic soluble (S) expression was considered favorable.
Table 2.
Mass Spec and SEC-LS Analysis of candidate uricases.
Table 3.
Differential scanning calorimetry stability comparison.
Fig 2.
Differential scanning calorimetry measurements.
Thermal stability was assessed for all candidate usicases by differential scanning calorimetry. Two transitions were observed for each uricase (TM1 and TM2). Deinococcus geothermalis uricase (A) has a low TM1 (32°C) whereas Deinococcus radiodurans uricase (B) has a TM1 of 55°C suggesting that Deinococcus geothermalis uricase is more thermally stable.
Table 4.
Enzymatic activity comparison (kcat and KM).
Fig 3.
Enzymatic activity comparison.
Substrate (UA) depletion assays were performed to assess uricase activity. The rate (specific activity) of UA oxidation was calculated based on a linear decrease in absorbance. Solid lines represent Michaelis-Menten kinetic fits performed in Prism GraphPad. Relative to Krystexxa®, Arthrobacter globiformis uricase had a 2 fold better kcat and Deinococcus geothermalis uricase had a 2 fold better KM (Table 4).
Fig 4.
The tripeptide, RGD has been shown to mediate cell adhesion through integrin binding. An M21 tumor cell adhesion assay was conducted to determine if the RGD motif in Arthrobacter globiformis uricase is surface accessible. M21 cells were used because they express αvβ3 and αvβ5 integrins. RGD-containing fibronectin substrate (positive control), Fab9mCys (an IgG that contains an RGD within the CDR-H3 loop), and uricase containing RGD all bind the M21 cells as measured by increase in fluorescence. However, PBS (negative control), and uricase containing the SGD had limited increase in fluorescence.
Fig 5.
Enzymatic activity comparison of RGD and SGD containing uricases.
Substrate (UA) depletion assays were performed to assess uricase activity. The rate (specific activity) of UA oxidation was calculated based on a linear decrease in absorbance. Solid lines represent Michaelis-Menten kinetic fits performed in Prism GraphPad. Both RGD and SGD containing Arthrobacter globiformis uricases appear to have comparable enzymatic activity.
Fig 6.
Ex vivo immunogenicity analysis.
The Epibase® assay is a human PBMC T-cell immunogenicity assay used to assess “immunogenicity risk”. In this assay, PBMC samples from 202 normal donors were used to screen the T-cell immunogenicity of a modified Arthrobacter globiformis uricase candidate relative to a negative control (buffer) and a positive control (KLH). The 202 donors were selected to represent HLA-DRB1 frequencies observed in a Caucasian population (S4 Fig). Stimulation indices (SI) values describe the ratio of proliferating CD3+CD4+ T-cell in antigen treated versus untreated wells. SI values >2 are considered positive which is supported by a p-value <0.05. A) Shows the individual SI values for negative control—0/202 donor samples (0%) responded with SI>2.0; and positive control—181/202 donors (91%) responded with a SI>2.0. B) Shows the individual SI values for negative control and modified uricase candidate—1/202 donor samples responded with a SI>2.0. C) Shows the mean stimulation index for all donor samples for buffer (SI = 1.0), KLH SI = 4.2 and modified Arthrobacter globiformis (SI = 1.03).
Fig 7.
PEGylation strategy and analysis.
(A) Shows the three dimensional solvent accessible sites within the tetrameric crystal structure of Arthrobacter globiformis uricase [1]. Each uricase monomer subunit of the tetrameric enzyme is shown in a different color, and residues selected for substitution with Cys (T11, N33, S142) are shown in yellow. (B) SDS-PAGE analysis of di-PEGylated and non-PEGylated uricase suggests that the di-PEGylated material runs at a much higher molecular weight relative to the non-PEGylated material. (C) A reverse-phase chromatography analysis of the di-PEGylated material suggests that 92.6% of the material is di-PEGylated, 4.4% mono-PEGylated, 0% unreacted and 2.7% over-PEGylated. (D) Demonstrates that the activity of di-PEGylated uricase is comparable to non-PEGylated uricase.
Fig 8.
In vivo rat PK and dog PK/PD assessment.
(A) In vivo rat PK comparison of non-PEGylated (blue triangles), di-PEGylated uricase (black circles) and tri-PEGylated uricase (green inverted triangles) and Krystexxa® (red squares). Rats were dosed IV at 5 mg/kg and samples were collected various time points and analyzed for residual uricase activity above background. Representative data from individual rats are shown. Both di-PEGylated and tri-PEGylated uricases have mono-phasic profiles. The half-life for the di-PEGylated uricase was ~22.8 hours and tri-PEGylated ~29.9 hours. Non-PEGylated uricase half-life in rats was 2–3 hours. Krystexxa® (red squares) had an atypical initial elimination profile followed by a relatively linear PK profile. (B) In vivo canine PK/PD study of di-PEGylated uricase delivered via SC route of administration at 3 mg/kg (blue squares) or 10 mg/kg (red squares). Hashed lines are associated with the right axis and represent the % UA measured in the blood.
Table 5.
In vivo rat PK comparison of di and tri-PEGylated uricases.
Fig 9.
Enzymatic activity analysis in human serum.
Substrate (UA) depletion assays were performed to assess uricase activity in 50% human serum. The rate (specific activity) of UA oxidation was calculated based on a linear decrease in absorbance. Solid (or dotted) lines represent Michaelis-Menten kinetic fits performed in Prism GraphPad. Di-PEGylated uricase activity was measured relative to Krystexxa®.