Immunogenicity-masking delivery of uricase against hyperuricemia and gout.

Improving the activity of uricase and lowering its immunogenicity remain significant challenges in the enzyme replacement management of hyperuricemia and related inflammatory diseases. Herein, an immunogenicity-masking strategy based on engineered red blood cells (RBCs) was developed for effective uricase delivery against both hyperuricemia and gout. The dynamic membrane of RBCs enabled high resistance to protease inactivation and hydrogen peroxide accumulation. Benefiting from these advantages, a single infusion of RBC-loaded uricase (Uri@RBC) performed prolonged blood circulation and sustained hyperuricemia management. Importantly, RBCs masked the immunogenicity of uricase, leading to the maintenance of UA-lowering performance after repeated infusion through reduced antibody-mediated macrophage clearance. In an acute gout model, Uri@RBC profoundly alleviated joint edema and inflammation with minimal systemic toxicity. This study supports the employment of immunogenicity-masking tools for efficient and safe enzyme delivery, and this strategy may be leveraged to improve the usefulness of enzyme replacement therapies for managing a wide range of inflammatory diseases.

Reducing Immunogenicity of Pegloticase With Concomitant Use of Mycophenolate Mofetil in Patients With Refractory Gout: A Phase II, Randomized, Double-Blind, Placebo-Controlled Trial.

OBJECTIVE: Pegloticase is used for the treatment of severe gout, but its use is limited by immunogenicity. This study was undertaken to evaluate whether mycophenolate mofetil (MMF) prolongs the efficacy of pegloticase. METHODS: Participants were randomized 3:1 to receive 1,000 mg MMF twice daily or placebo for 14 weeks, starting 2 weeks before receiving pegloticase and continuing while receiving intravenous pegloticase 8 mg biweekly for 12 weeks. Participants then received pegloticase alone from week 12 to week 24. The primary end points were the proportion of patients who sustained a serum urate level of ≤6 mg/dl at 12 weeks and the rate of adverse events (AEs). Secondary end points included 24-week durability of serum urate level ≤6 mg/dl. Fisher's exact test and Wilcoxon's 2-sample test were used for analyses, along with Kaplan-Meier estimates and log rank tests. RESULTS: A total of 32 participants received ≥1 dose of pegloticase. Participants were predominantly men (88%), with a mean age of 55.2 years, mean gout duration of 13.4 years, and mean baseline serum urate level of 9.2 mg/dl. At 12 weeks, a serum urate level of ≤6 mg/dl was achieved in 19 (86%) of 22 participants in the MMF arm compared to 4 (40%) of 10 in the placebo arm (P = 0.01). At week 24, the serum urate level was ≤6 mg/dl in 68% of MMF-treated patients versus 30% of placebo-treated patients (P = 0.06), and rates of AEs were similar between groups, with more infusion reactions occurring in the placebo arm (30% versus 0%). CONCLUSION: Our findings indicate that MMF therapy with pegloticase is well tolerated and shows a clinically meaningful improvement in targeted serum urate level of ≤6 mg/dl at 12 and 24 weeks. This study suggests an innovative approach to pegloticase therapy in gout.

Structure-Based Immunogenicity Prediction of Uricase from Fungal (Aspergillus flavus), Bacterial (Bacillus subtillis) and Mammalian Sources Using Immunoinformatic Approach.

Gout is a common rheumatic condition caused due to increase in serum uric acid level (hyperuricemia). Uricase is for lowering the level of uric acid but unfortunately, it is not produced in humans due to evolutionary changes. Therefore, it is administered to humans from outside in case of the high uric acid level in blood. A different formulation of uricase from bacterial, fungal, and mammalian sources is present in the market for the treatment of hyperuricemia conditions. Uricase formulation showed immunogenic response due to the occurrence of hypersensitivity reaction during the treatment that results in poor patient compliance. The purpose of this study was to clarify the variation of Uricase immunogenicity from different sources. We have used some immunoinformatic approaches to analyze and understand some structural aspects of immunogenic and allergenic epitopes of Uricase by calculation of relative frequency for eleven global alleles. As per our knowledge, this is the first immunoinformatic study of Uricase (structural based immunogenicity prediction) that deciphered the high immunogenic nature of Uricase but no significant difference in immunogenicity was found among Uricase isolated from Aspergillus flavus, Bacillus subtillis, and mammalian source. This study gives a further lead to develop some methods (include bioengineering of less immunogenic version of the uricase or utilizing the homologous enzymes) for minimizing immune response or search new sources of uricase that could be less or non-immunogenic.

Species and sex differences in the blood clearance and immunogenicity of PEGylated uricase: A comparative 26-week toxicity study in rats and monkeys.

Low response rates and high immunogenicity were observed after repeated injections of pegloticase (Krystexxa) into gout patients during clinical trials. However, related research had not been reported in preclinical animal experiments, which has limited the development of this drug. In this study, the toxicity of mPEG-UHC was studied in rats and monkeys over a 26-week period of repeated intravenous dosing. There were no obvious toxic reactions in the tested animals, with the exception of mPEG-UHC blood clearance and immunogenicity. After repeated injections of mPEG-UHC, rapid loss of uricolytic activity (RLA) was not detected in rats, whereas RLA was observed in 44.4% of drug-treated monkeys. In these monkeys, RLA was observed in 11.1% of males and 77.8% of females, and such incidences increased with higher dosing. High titres of anti-uricase IgG antibodies were associated with RLA but did not result in any toxicity. Remission and recurrence of RLA occurred in one female monkey in the high-dose group because of suppressed and altered immune responses in this animal. The predicted incidence of RLA after repeated injections of mPEG-UHC in gout patients may be lower than that of pegloticase. In this study, the no-observed-adverse-effect levels (NOAELs) of mPEG-UHC in rats and monkeys were 32.0 mg/kg and 20.0 mg/kg, respectively. Therefore, the results showed that rats and monkeys could tolerate long-term and high-dose administrations of mPEG-UHC, and mPEG-UHC blood clearance and immunogenicity showed obvious species and sex differences. These findings will provide valuable information to direct the clinical use of mPEG-UHC.

A minimal physiologically based pharmacokinetic model that predicts anti-PEG IgG-mediated clearance of PEGylated drugs in human and mouse.

Circulating antibodies that specifically bind polyethylene glycol (PEG), a polymer routinely used in protein and nanoparticle therapeutics, have been associated with reduced efficacy and increased adverse reactions to some PEGylated therapeutics. In addition to acute induction of anti-PEG antibodies (APA) by PEGylated drugs, typically low but detectable levels of APA are also found in up to 70% of the general population. Despite the broad implications of APA, the dynamics of APA-mediated clearance of PEGylated drugs, and why many patients continue to respond to PEGylated drugs despite the presence of pre-existing APA, remains not well understood. Here, we developed a minimal physiologically based pharmacokinetic (mPBPK) model that incorporates various properties of APA and PEGylated drugs. Our mPBPK model reproduced clinical PK data of APA-mediated accelerated blood clearance of pegloticase, as well as APA-dependent elimination of PEGyated liposomes in mice. Our model predicts that the prolonged circulation of PEGylated drugs will be compromised only at APA concentrations greater than ~500 ng/mL, providing a quantitative explanation to why the effects of APA on PEGylated treatments appear to be limited in most patients. This mPBPK model is readily adaptable to other PEGylated drugs and particles to predict the precise levels of APA that could render them ineffective, providing a powerful tool to support the development and interpretation of preclinical and clinical studies of various PEGylated therapeutics.

Polypeptides with High Zwitterion Density for Safe and Effective Therapeutics.

The commonly used "stealth material" poly(ethylene glycol) (PEG) effectively promotes the pharmacokinetics of therapeutic cargos while reducing their immune response. However, recent studies have suggested that PEG could induce adverse reactions, including the emergence of anti-PEG antibodies and tissue histologic changes. An alternative stealth material with no or less immunogenicity and organ toxicity is thus urgently needed. We designed a polypeptide with high zwitterion density (PepCB) as a stealth material for therapeutics. Neither tissue histological changes in liver, kidney, or spleen, nor abnormal behavior, sickness or death was induced by the synthesized polymer after high-dosage administration for three months in rats. When conjugated to a therapeutic protein uricase, the uricase-PepCB bioconjugate showed significantly improved pharmacokinetics and immunological properties compared with uricase-PEG conjugates.

A Therapeutic Uricase with Reduced Immunogenicity Risk and Improved Development Properties.

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.

Potential induction of anti-PEG antibodies and complement activation toward PEGylated therapeutics.

Conjugation of polyethylene glycol (PEG) to therapeutics has proven to be an effective approach to increase the serum half-life. However, the increased use of PEGylated therapeutics has resulted in unexpected immune-mediated side-effects. There are claims that these are caused by anti-PEG antibodies inducing rapid clearance. These claims are however hampered by the lack of standardized and well-validated antibody assays. PEGylation has also been associated with the activation of the complement system causing severe hypersensitivity reactions. Here, we critically review the clinical and analytical tools used. In addition, we propose an explanation of the immune-mediated side-effects of PEGylated products based on the haptogenic properties of PEG, responsible for complement activation and the induction of anti-PEG antibodies.

PEG-ing down (and preventing?) the cause of pegloticase failure.

Pegloticase is a powerful but underutilized weapon in the rheumatologist's armamentarium. The drug's immunogenicity leads to neutralizing antibody formation and rapid loss of efficacy in roughly one-half of all patients, which remains an impediment to broader use. New data, however, suggest that drug survival might improve with concomitant immunosuppressive agent (s), which merits further study. Efficacy appears to be unchanged when pegloticase is infused at 3-week (rather than 2-week) intervals. Stretching the time between infusions may also improve patient adherence and allow for earlier identification of transient responders.

Induced and pre-existing anti-polyethylene glycol antibody in a trial of every 3-week dosing of pegloticase for refractory gout, including in organ transplant recipients.

INTRODUCTION: Pegloticase, a PEGylated recombinant porcine uricase, is approved for treating refractory gout at a dose of 8 mg intravenous (IV) every 2 weeks. However, during phase 1 testing, pharmacokinetics supported less frequent dosing. Also, single doses of pegloticase unexpectedly induced antibodies (Ab) that bound to polyethylene glycol (PEG). We have conducted a phase 2 trial to evaluate every 3-week dosing, and to further define the Ab response to pegloticase. Organ transplant recipients were included, as they are prone to severe gout that is difficult to manage, and because treatment to prevent graft rejection might influence the immune response to pegloticase. METHODS: Plasma uricase activity (pUox), urate concentration (pUA), and clinical response were monitored during up to 5 infusions in 30 patients, including 7 organ transplant recipients. Depending on whether pUA <6 mg/dL was achieved and maintained, patients were classified as non (NR), persistent (PR), or transient (TR) responders. Ab to pegloticase and 10 kDa mPEG were monitored by enzyme linked immunosorbent assay and specificity was further defined. RESULTS: We observed 17 PR, 12 TR, and 1 NR; 21 patients (16 PR, 5 TR) received all 5 infusions. Over the 15-week trial, pUA in PR averaged 1.0 ± 0.4 mg/dL; T½ for pUox was approximately 13 days, and area under the curve after dose 5 was approximately 30% higher than after dose 1. PR showed clinical benefit and in some, tophi resolved. In 11 of 12 TR, pUox fell rapidly and hyperuricemia recurred before dose 2. In all TR and NR, loss of response to pegloticase was accompanied by Ab to PEG, which was pre-existing in half of those who had no prior exposure to pegloticase. No PR, and 1 one out of 7 organ transplant recipients, had a sustained Ab response to pegloticase. CONCLUSIONS: Every 3-week dosing is effective and may enhance the utility of pegloticase for treating refractory gout. Ab to PEG, which were pre-existing or induced by treatment, caused rapid loss of efficacy and increased the risk of infusion reactions. Organ transplant recipients can benefit from pegloticase, and may be less prone than non-recipients to developing anti-PEG Ab. Investigation of immunosuppressive strategies to minimize anti-PEG Ab is warranted. TRIAL REGISTRATION: ClincalTrials.gov identifier: NCT00111657.

Pegloticase immunogenicity: the relationship between efficacy and antibody development in patients treated for refractory chronic gout.

INTRODUCTION: The efficacy of pegloticase, a polyethylene glycol (PEG)-conjugated mammalian recombinant uricase, approved for chronic refractory gout, can be limited by the development of antibodies (Ab). Analyses from 2 replicate, 6-month, randomized controlled trials were performed to characterize Ab responses to pegloticase. METHODS: Anti-pegloticase, anti-PEG, and anti-uricase Ab were determined by validated enzyme-linked immunosorbent assays. Ab titers were analyzed for possible relationships with serum pegloticase concentrations, serum uric acid (sUA) lowering, and risk of infusion reactions (IRs). RESULTS: Sixty-nine (41%) of 169 patients receiving pegloticase developed high titer anti-pegloticase Ab (> 1:2430) and 40% (67/169) developed anti-PEG Ab; 1 patient receiving placebo developed high titer anti-pegloticase Ab. Only 14% (24/169) of patients developed anti-uricase Ab, usually at low titer. In responders, patients showing sustained UA lowering, mean anti-pegloticase titers at week 25 (1:837 ± 1687 with biweekly and 1:2025 ± 4506 with monthly dosing) were markedly lower than in nonresponders (1:34,528 ± 42,228 and 1:89,658 ± 297,797, respectively). Nonresponder status was associated with reduced serum pegloticase concentrations. Baseline anti-pegloticase Ab, evident in 15% (31/212) of patients, did not predict subsequent loss of urate-lowering response. Loss of sUA response preceded IRs in 44 of 56 (79%) pegloticase-treated patients. CONCLUSIONS: Loss of responsiveness to pegloticase is associated with the development of high titer anti-pegloticase Ab that increase clearance of pegloticase and are associated with a loss of the sUA lowering effect and increased IR risk. Pre-infusion sUA can be used as a surrogate for the presence of deleterious anti-pegloticase Ab. TRIAL REGISTRATION: NCT00325195. Registered 10 May 2006, NCT01356498. Registered 27 October 2008.

[Preparation and characterization of recombinant protein A affinity packing].

To obtain an excellent antibody purification medium, affinity chromatographic packing with recombinant staphylococcal protein A (rProtein A) was synthesized and verified. With E. coli cells harboring the recombinant plasmid, the rProtein A was expressed and purified, then was conjugated to epichlorohydrin-activated Sepharose 4 Fast Flow to prepare an affinity chromatographic packing. The performances of the packing were validated with rabbit antiurate oxidase. After the reaction, the concentration of rProtein A coupled to Sepharose 4 Fast Flow was 1.5 x 10(-4) mol/L. Scatchard analysis of the binding isotherm for IgG showed excellent binding capacity on the adsorbent, giving a dissociation constant (Kd) of 2.28 x 10(-7) mol/L and a theoretical maximum adsorption capacity of 20. 697 g/L. The identification showed the packing was stable in 0.1 mol/L NaOH solution at 1 h. By using the packing, the pure antibody exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from rabbit serum after one-step elution, with 96. 1% of yield and 19 mg IgG for one milliliter of gel. The research laid the foundation of the localization of rProtein A affinity packing.

Egg yolk and serum antibody titers of broiler breeder hens immunized with uricase and or urease.

This study evaluated whether broiler breeder hens immunized with uricase (UC), urease (UE), or UC + UE would develop antibody (IgY) titers against these enzymes to prevent manure-N degradation and NH(3) release. Ross × Arbor Acres hens were assigned to PBS (control), UC, UE, or UC + UE injection treatments. Each group had 19 hens per treatment. On d 0, each of the enzymes or PBS was emulsified with complete Freund's adjuvant and administered intramuscularly, whereas on d 7 and 14, a booster injection of PBS or enzymes was administered as an incomplete adjuvant. Blood samples were taken on d 0, 4, 9, 12, 17, 21, and 24 for serum-specific IgY titer analysis. Eggs were collected for yolk-specific IgY titer analysis. Manure samples were taken for nutrient, pH, and NH(3) measurements. Elevated egg yolk anti-UC-IgY titers were observed from UC-immunized hens after the second immunization (P ≤ 0.0001), and they remained higher than those of the PBS- or UE-immunized hens from d 9 to 24. After the first injection, egg yolk anti-UE-IgY titers from hens immunized with UE or the combined antigen were greater than those of birds injected with PBS or UC (P ≤ 0.01). The serum anti-UC-IgY response to UC immunization was observed after the first injection (P ≤ 0.01) and on d 9 (P ≤ 0.0001), and titers remained greater than those of hens immunized with PBS or UE until d 28. The serum anti-UE-IgY titers remained low until much later compared with the anti-UC-IgY titers. Only at 24 and 28 d were anti-UE-IgY titers significantly greater in the UE-immunized hens than in hens immunized with PBS or UC. Hens immunized with UC or UE responded with both egg yolk and serum IgY titers. The combined antigens were significantly greater than the PBS control but had less effect than the individual UC or UE in both the egg yolk and serum. These findings indicate that despite measurable egg yolk and serum IgY titers, immunizing hens with UC, UE, or the combined antigens did not affect the manure nutrients or NH(3) emissions of the treated hens.

Designing and engineering of a site-specific incorporation of a keto group in uricase.

Urate oxidase is a potential therapeutic protein in the prevention and treatment of tumor lysis syndrome and hyperuricemia. However, its severe immunogenicity limits its clinical application. In our work, several strides have been made toward engineering site-specific modifications of keto groups in urate oxidase by using evolved Methanocaldococcus jannaschii aminoacyl-tRNA synthetase(s)/suppressor tRNA pairs to reduce its antigenicity. Our approach, described here, consisted of designing a M. jannaschii tyrosyl-tRNA synthetase library based on the homology modeling and molecular docking model of the species-specific TyrRS-Tyr complex. The active mutation was picked, and pBR-RS series vectors were constructed to define the relationship between the expression of aaRS and the efficiency of the orthogonal amber suppressor tRNA/synthetase system. Two sites based on the 3D structure of the Candida utilis uricase, Lys21 and Lys248, were substituted for p-acetyl-l-phenylalanine, and the yields were optimized. The products were purified, and their enzyme activities and antigenic properties were analyzed. The mutated uricase exhibited decreased antigenic properties, while its catalytic activities remained unchanged. This method imparts new insights into structure-function relationship research and provides a means by which site-specific modifications may be achieved by using PEG derivates to improve pharmacological properties of urate oxidase.

Biochemical and biopharmaceutical properties of PEGylated uricase.

PEGylation is a successful strategy for improving the biochemical and biopharmaceutical properties of proteins and peptides through the covalent attachment of polyethylene glycol chains. In this work, purified recombinant uricase from Candida sp. (UC-r) was modified by PEGylation with metoxypolyethilenoglycol-p-nitrophenyl-carbonate (mPEG-pNP) and metoxypolyethyleneglycol-4,6-dichloro-s-triazine (mPEG-CN). The UC-r-mPEG-pNP and UC-r-mPEG-CN conjugates retained 87% and 75% enzyme activity respectively. The K(M) values obtained 2.7x10(-5) M (mPEG-pNP) or 3.0x10(-5) M (mPEG-CN) for the conjugates as compared to 5.4x10(-5) M for the native UC-r, suggesting enhancement in the substrate affinity of the enzyme attached. The effects of pH and temperature on PEGylated UC-r indicated that the conjugates were more active at close physiological pH and were stable up to 70 degrees C. Spectroscopic study performed by circular dichroism at 20 degrees C and 50 degrees C did not show any relevant difference in protein structure between native and PEGylated UC-r. In rabbit and Balb/c mice, the native UC-r elicited an intense immune response being highly immunogenic. On the other hand, the PEGylated UC-r when injected chronically in mice did not induce any detectable antibody response. This indicates sufficient reduction of the immunogenicity this enzyme by mPEG-pNP or mPEG-CN conjugation, making it suitable for a possible therapeutical use.

Pharmacokinetics and pharmacodynamics of intravenous PEGylated recombinant mammalian urate oxidase in patients with refractory gout.

OBJECTIVE: To evaluate the efficacy, immunogenicity, and tolerability of intravenous (IV) PEGylated recombinant mammalian urate oxidase (PEG-uricase) for the treatment of severe gout. METHODS: Single infusions of PEG-uricase (at doses ranging from 0.5 mg to 12 mg) were administered to 24 patients (6 cohorts of 4 patients each) in a phase I clinical trial. Plasma uricase activity (pUox), the plasma urate concentration (pUAc), and the uric acid-to-creatinine ratio (UAc:Cr) in urine were monitored for 21 days after dosing. Adverse events and the IgG antibody response to PEG-uricase were followed up for 35 days. RESULTS: All patients completed the trial. Maximum pUox was linearly related to the IV dose of PEG-uricase, the area under the curve (AUC) value increased linearly (up to a dose of 8 mg), and the pUox half-life was 6.4-13.8 days. After doses of 4-12 mg, the pUAc fell within 24-72 hours, from a mean +/- SD value of 11.1 +/- 0.6 mg/dl to 1.0 +/- 0.5 mg/dl; the AUC value for the pUAc was equivalent to maintaining the pUAc at 1.2-4.7 mg/dl for 21 days postinfusion. The UAc:Cr ratio in urine fell in parallel with the pUAc. IgG antibodies to PEG-uricase, mostly IgG2 and specific for PEG, developed in 9 patients, who had more rapid enzyme clearance but no allergic reactions. All adverse events were mild to moderate, with gout flares being most common. CONCLUSION: The bioavailability, efficacy, and tolerability of IV PEG-uricase were greater than the bioavailability, efficacy, and tolerability observed in a previous phase I trial of subcutaneous PEG-uricase. Infusing 4-12 mg of PEG-uricase every 2-4 weeks should maintain the pUAc well below the therapeutic target of 6 mg/dl and greatly reduce renal uric acid excretion. This treatment could be effective in depleting expanded tissue urate stores in patients with chronic or tophaceous gout.

Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase.

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 +/- 2.1 mg/dl (mean +/- SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 +/- 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.

Production of an egg yolk antibody specific to microbial uricase and its inhibitory effects on uricase activity.

Ammonia gas produced from poultry manure can be a potential source of environmental pollution. Microbial uricase in poultry manure is an important target enzyme to reduce ammonia production because ammonia is mainly generated from the microbial decomposition of uric acid in the manure. Thus, the inhibition of microbial uricase is critical in preventing NH3 volatilization. A potential method of inhibiting uricase activity is the use of antibodies specific to microbial uricase. Therefore, this study was designed to evaluate 1) the production of the uricase-specific egg yolk antibody (IgY) from immunized hens and 2) the effect of the uricase-specific IgY on the activity of uricase. A total of 12 Single Comb White Leghorn hens were injected intramuscularly with uricase from Arthrobacter globiformis. The hens were immunized a second and third time at 1 and 2 wk, respectively, after the initial injection. The production of uricase-specific IgY was first detected at 2 wk after the initial immunization, and levels increased more than threefold at 4 wk. The method including water extraction, ammonium sulfate precipitation, and ethanol precipitation showed the most acceptable IgY purity and over 97% uricase-specific IgY recovery. Finally, the effect of the uricase-specific IgY on uricase activity was determined by a uricase assay. The slope ratio showed that the immune-IgY from the uricase-immunized hens and nonimmune IgY from nonimmunized control hens reduced uricase degradation by 58 and 43%, respectively, compared to the uricase treatment. The regression slope indicated that the immune IgY and nonimmune IgY treatments had a significant inhibitory effects on uricase activity compared to the uricase.

Altered antigenic disposition of peroxisomal urate oxidase in PEX5-defective Chinese hamster ovary cells.

Since Chinese hamster ovary (CHO) cells never express urate oxidase (UO), we tried to establish cell lines stably producing UO in order to elucidate the peroxisomal import process. The enzyme is a peroxisome targeting signal 1 (PTS1) protein harboring SKL motif at the carboxy-terminus [Biochem. Biophys. Res. Commun. 158 (1989) 991] and PEX5 protein (Pex5p) is supposed to be involved in the import process [Nat. Genet. 9 (1995) 115; J. Cell Biol. 130 (1995) 51]. We transfected a cDNA encoding rat UO into both wild type and PEX5-defective CHO cells to isolate each cell line stably producing the enzyme. While we examined the import process of UO in mutant cells, we noticed an interesting observation by using polyclonal antibody U1 or U2, which separately recognizes epitopes of UO. U1 antibody mainly interacts with epitopes in the amino-terminal region of UO. On the other hand, U2 antibody reacts with many epitopes distributed in the broad region of UO molecule. When UO produced in cultured cells was stained with U2 antibody, the enzyme was detected in peroxisomes of both wild type and PEX5-mutant cells. Whereas, U1 antibody stained the peroxisomal UO in wild type cells, but not in PEX5-mutant cells. These immunocytochemical observations suggest that the epitopes at the amino-terminal region of UO will be concealed in mutant cells. When the mutant cells were transfected with wild type PEX5 cDNA, U1 antibody came to react with UO in peroxisomes of mutant cells. The restoration indicates that the exposure of N-terminal epitopes of UO will depend upon the functional Pex5p. Immunoelectron microscopic observation showed that the peroxisomal import of UO was partially retarded in PEX5 mutant cells. The observation also supported the fact that UO was mainly localized in the peroxisomal matrix of wild type cells but in the membrane of mutant cells.