PEGylation Confers Greatly Extended Half-Life and Attenuated Immunogenicity to Recombinant Methioninase in Primates

INTRODUCTION

Methionine dependence, the elevated minimal methionine requirement for cell growth relative to normal cells, has been observed in many human cancer cell lines and cancer xenografts in animal models (1, 2, 3) . Methionine dependence is a metabolic defect seen only in cancer cells and precludes the cells from growing in medium in which methionine is depleted (4 , 5) .

l-methionine α-deamino-γ-mercaptomethane lyase (methioninase, METase), is a pyridoxal-l-phosphate (PLP)-dependent enzyme that cleaves methionine. METase has been demonstrated to be a powerful approach to methionine depletion in vivo (6 , 7) . The enzyme has been cloned from Pseudomona putida and produced in Escherichia coli (refs. 8 , 9 ; recombinant methioninase, rMETase) for extensive preclinical testing.

rMETase alone or in combination with chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) have shown efficacy and synergy, respectively, in mouse models of colon cancer, lung cancer, and brain cancer (10, 11, 12, 13) . The findings from a pilot Phase I clinical trial showed that METase depleted plasma methionine levels without observed clinical toxicity over a period up to ∼24 hours in patients with advanced cancer (14 , 15) .

To determine the long-term effects of methionine depletion mediated by repeated administration of rMETase, a 2-week i.v. administration of 4,000 units/kg of rMETase every 8 hours in macaque monkeys was carried out previously (16) . This regimen resulted in a steady-state depletion of plasma methionine to <2 μmol/L. rMETase was eliminated with a biological half-life (T_1/2) of 2.5 hours. The only manifest toxicity was decreased food intake and slight weight loss. Serum albumin and red cell values declined transiently during treatment. However, challenge on day 28 resulted in severe anaphylactic shock and death in 1 animal, although subsequent pretreatment with hydrocortisone prevented the anaphylactic reaction when rechallenge was carried out at days 66, 86, and 116. Significant anti-rMETase antibody titers were found up to 10⁻⁶ after the fourth challenge. These results indicated that the therapeutic potential of rMETase was limited by its short plasma circulating half-life and potentially severe immunologic reactions.

Conjugation of protein therapeutics with polyethylene glycol (PEG) has been shown to confer important therapeutic benefits, most importantly reduced antigenicity (17) . The Food and Drug Administration has approved the PEGylated forms of several protein therapeutics for clinical use including adenosine deaminase, asparaginase, α-interferon, and a growth hormone antagonist (18, 19, 20, 21, 22) .

To improve the rMETase therapeutic potential, rMETase was coupled to methoxypolyethylene glycol succinimidyl glutarate-5000 (MEGC-PEG-5000; ref. 23 ). Pharmacokinetic evaluation in mice showed that MEGC-PEG-rMETase increased the serum half-life of the enzyme up to 20-fold and increased methionine depletion time up to 12-fold compared with unmodified rMETase. In addition, a further prolongation of in vivo activity and effective methionine depletion by MEGC-PEG-rMETase was achieved by the simultaneous administration of pyridoxal-5′-phosphate (PLP; ref. 23 ).

To additionally investigate the clinical potential of MEGC-PEG-rMETase, the current study investigated the pharmacokinetics, antigenicity, and systemic toxicity of repeated administration of MEGC-PEG-rMETase in macaque monkeys.

MATERIALS AND METHODS

MEGC-PEG-rMETase.

MEGC-PEG-rMETase was prepared by Shionogi and Co. Ltd. (Osaka, Japan) based on a method described previously (23) . MEGC-PEG-5000 was coupled to rMETase using molar ratios of PEG/rMETase of 60:1. Analysis with matrix-assisted desorption ionization-time of flight/ mass spectrometry showed that each MEGC-PEG-rMETase subunit has a 59.8 to 81.0 kDa molecular mass, corresponding to 3 to 7 PEG molecules per subunit. The protein content of MEGC-PEG-rMETase was 39.9 mg/mL. The specific enzyme activity was 44 units/mg. Frozen MEGC-PEG-rMETase was thawed and warmed to 37°C just before use. The vehicle solution was constituted with 10 mmol/L sodium phosphate buffer containing 0.1 mmol/L PLP (pH 7.9).

Animals.

Male Macaca fascicularis monkeys, ages 5 to 7 years, and weighing 6 to 8 kg, were bred and used for this study at the Suzhou West Hill Experimental Animal Co., Ltd. (Suzhou, People’s Republic of China). The monkeys were individually housed in stainless steel cages in a controlled environment (12 hours/day artificial lighting; 12 times/hour ventilation; 20°C to 25°C room temperature; 40 to 60% humidity). The monkeys were fed monkey chow, fruit, and water at scheduled times. All of the monkeys were acclimated in their cages for a 2-week period before the initiation of treatment. During this period, the monkeys were observed for clinical signs and examined for food consumption, body weight, hematology, and blood chemistry.

Dose Ranging.

A dose-escalating strategy used 1,000 units/kg, 4,000 units/kg, and 8,000 units/kg MEGC-PEG-rMETase administered i.v. to the same monkeys via the saphenous veins of the hind limbs. A 2-week interval was observed between each dose. The monkeys were fasted 8 hours before and after dosing. Blood samples were collected at different time points after each dosing and measured for plasma rMETase activity and methionine levels.

Dose and Challenge Schedule.

On the basis of the dose-ranging study, two naïve monkeys (monkeys 1 and 2) were administered MEGC-PEG-rMETase at a dose of 4,000 units/kg each, twice daily at 12-hour intervals for 7 consecutive days. As an untreated control, one monkey (monkey 3) received the same volume of vehicle solution in the same manner as the MEGC-PEG-rMETase-treated monkeys. On days 29, 50, and 71 after administration of MEGC-PEG-rMETase, the two MEGC-PEG-rMETase-treated monkeys were given challenge injections of MEGC-PEG-rMETase at a dose of 4,000 units/kg. All of the animals were fasted 2 hours before and after each injection.

Determination of Plasma rMETase Activity.

During the 7-day repeated dosing and challenge injections of MEGC-PEG-rMETase, blood samples were drawn on days 1, 4, 7, 29, 50, and 71 at time points before the first rMETase injection of the day and at 1 hour, 2 hours, 4 hours, 8 hours, 10 hours, and 12 hours after injection on each sampling day. All of the blood samples were collected in EDTA-coated tubes and centrifuged at 13,000 rpm for 2 minutes at 4°C to obtain plasma. Plasma samples were aliquoted and frozen at −70°C until analysis for plasma rMETase activity and methionine concentration.

The amount of MEGC-PEG-rMETase apoenzyme was determined from α-ketobutyrate produced from l-methionine in the presence of PLP according to the method of Esaki and Soda (24) with slight modification (25) . Fifty microliters of sample diluted in 100 mmol/L potassium phosphate buffer (pH 8.0) containing 0.01% DTT, 1 mmol/L EDTA₂Na, 10 μmol/L PLP, and 0.05% Tween 80 was mixed with 1 mL of substrate solution [100 mmol/L potassium phosphate buffer (pH 8.0) containing 25 mmol/L l-methionine and 10 μmol/L PLP] in a glass test tube. The reaction mixture was vortexed immediately and incubated at 37°C without shaking for precisely 10 minutes. The reaction was stopped by adding 100 μL 50% trichloroacetic acid. The suspension was centrifuged at 13,000 rpm for 2 minutes. The supernatant (0.8 mL) was collected in a glass tube containing 1.6 mL of 1 mol/L acetate buffer (pH 5.0). Then, 0.6 mL of 3-methyl-2-benzothiazolinone hydrazone solution containing 0.1% 3-methyl-2-benzothiazolinone hydrazone dydrochoride monohydrate (Wako, Osaka, Japan) was added to the tube, mixed well, and incubated at 50°C for 40 minutes. The absorbance of the reaction mixture was measured at 320 nm. The assay was carried out in triplicate. ΔE was calculated by subtracting the average absorbance of blanks from the average absorbance of the reaction mixture. The enzyme activity was calculated by the following formula: Activity (units/mL) = 0.548 (1.07 + 2.2ΔE) ΔE. One unit of enzyme is defined as the amount of enzyme that produced 1 μmol/L of α-ketobutyrate per minute at an infinite concentration of l-methionine.

The amount of MEGC-PEG-rMETase holoenzyme was determined in the absence of PLP in the substrate solution. All of the procedures were the same as the PEG-rMETase apoenzyme assay except that 5 μL of the sample in 45 μL of distilled water was mixed with 1 mL of substrate solution in the first reaction step. Due to the change of sample volume, enzyme activity was calculated from the following formula: Activity (units/mL) = 27.4 (1.07 + 2.2ΔE) ΔE.

Determination of Plasma Methionine.

The plasma methionine concentration was measured using a precolumn derivatization, followed by high-performance liquid chromatography separation based on a method described previously with modification (26) . Ten microliters of plasma sample or methionine standard was used. The plasma methionine was identified by the retention time of a methionine standard solution and quantitated according to a methionine standard curve. The limit of detection was 0.5 μmol/L methionine. Briefly, 10 μL of plasma sample or methionine standard was precipitated with 30 μL of acetonitrile, followed by centrifugation at 10,000 rpm for 5 minutes. Ten microliters of the supernatant was mixed with 5 μL of a fluoraldehyde derivative reagent, O-phthaldialdehyde, for 1 minute at room temperature, followed by addition of 150 μL of 0.1 mol/L sodium acetate (pH 7.0). Twenty microliters of the reaction mixture were loaded on a reversed-phase Supelcosil LC-18DB column (25 cm × 4.8 cm, particle size 5 μm, Supelco, Bellefonte, PA). The amino acid derivatives were separated by using a gradient elution of 40 to 60% solution B (Methanol) in solution A [tetrahydrofuran/methanol/0.1 mol/L sodium acetate (pH 7.2), 5:95:900] at a flow rate of 1.5 mL/minute. A fluorescence spectrophotometer was used for detection, with excitation at 350 nm and emission at 450 nm. The plasma methionine was identified by the retention time of a methionine standard solution and quantitated according to a methionine standard curve (Table 1) ⇓ .

Pharmacokinetic Analysis.

The concentration-time curves for plasma MEGC-PEG-rMETase apoenzyme and holoenzyme were generated as the plasma enzyme activity (units/mL) versus time after a single MEGC-PEG-rMETase administration at doses of 2,000 units/kg and 4,000 units/kg (Table 2) ⇓ . An appropriate pharmacokinetic model was chosen on the basis of the profile of the concentration-time curve. Pharmacokinetics parameters including initial drug concentration, elimination constant, apparent volume of distribution, T_1/2, area under (concentration–time) curve (AUC), and total body clearance (CL) were calculated for a one-compartment model (Table 2) ⇓ . Parameters including apparent volume of distribution, distributive-phase half-life, elimination-phase half-life, rate constant for exchange between the peripheral and central compartment, rate constant for exchange between the central and peripheral compartment, total elimination rate constant, AUC, and CLs were calculated for a two-compartment model (Table 2) ⇓ . Pharmacokinetic modeling and pharmacokinetic parameter estimations were carried out using pharmacokinetic software 3P97 (Mathematics Institute, Academy of Science, Beijing, China).

Systemic Toxicity Studies.

Clinical parameters including vital signs, gastrointestinal symptoms, infections, and overall activity and behavior were observed daily. Food consumption and body weights were recorded daily during the treatment period. Average daily food consumption of each week was calculated weekly.

Hematology and Blood Chemistry.

For hematology and blood chemistry examinations, 2-mL blood samples were collected via the saphenous vein from each animal. Collections were made on days 3, 6, 11, 22, 29, 33, 50, 54, 71, and 75 during the experimental period. Approximately 1 mL of the blood sample was collected into EDTA-coated tubes. Blood was analyzed with an automated hematology analyzer for the following hematologic parameters: red blood cells, white blood cells, platelets, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin concentration (Table 3) ⇓ . Approximately 1 mL of the blood sample was collected into serum separator tubes and centrifuged. The resulting sera samples were analyzed with an automated chemistry analyzer for the following blood chemistry parameters: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, cholesterol, glucose, blood urea nitrogen, creatinine, Ca2+, NA+, K+, and CI− (Table 3) ⇓ .

Histopathology.

One monkey from the MEGC-PEG-rMETase-treated group and the control monkey were sacrificed at the end of the study. Gross necropsy was performed. Brain, lung, liver, adrenals, kidneys, and spleen were weighed. The tissues from the heart, aorta, lungs, liver, and kidneys were immediately collected and fixed in 10% neutral-buffered formalin for histopathology examinations.

Determination of Plasma Anti-rMETase Antibody.

Blood samples were collected on days 3, 6, 29, 33, 50, 54, 71, and 75 after administration of MEGC-PEG-rMETase for determination of plasma anti-rMETase antibody (Table 4) ⇓ . Plasma was prepared and stored at −70°C. Plasma anti-PEG-rMETase IgG antibody measurements were performed using a sandwich ELISA as described previously (23) . 100 μL rMETase (200 μg/ml) in 0.1 mol/L carbonate coating buffer (pH 9.5) were added to each well of a 96-well microplate and incubated at 4°C overnight. The plate was washed three times with PBS (pH 7.4) containing 0.05% Tween 20, blocked for 2 hours at room temperature with 200 μL PBS assay buffer (pH 7.4) containing 10% FBS. After washing three times, 100 μL of 10-fold serial dilutions of the plasma samples in PBS were added to appropriate wells and incubated for 2 hours at room temperature, followed by washing again. One hundred microliters of optimally diluted antimonkey IgG immunoglobulin and horseradish peroxidase conjugate (Sigma, St. Louis, MO) were added to each well. The plate was incubated for 1 hour at room temperature and washed three times. One hundred microliters of substrate solution containing O-phenylenediamine dihydrochloride and hydrogen peroxide (Sigma) were added to each well, followed by 30-minute incubation at room temperature. Fifty microliters of 2 N sulfuric acid were added to each well to stop the color reaction, and the absorbance of each well was measured at 492 nm.

The presence of neutralizing antibody was examined in plasma (0.2 mL) obtained from rMETase-treated monkeys. This plasma was mixed with 0.2 mL of various concentrations of rMETase solution. The saline and plasma obtained from a vehicle-treated monkey were used as blank and negative control, respectively. After incubation at 37°C in a water bath for 30 minutes, the reaction mixtures were measured for rMETase enzyme activity.

RESULTS AND DISCUSSION

Dose-Ranging Study for Optimal MEGC-PEG-rMETaseMediated Methionine Depletion.

Plasma methionine depletion was determined in the monkeys after three single doses of MEGC-PEG-rMETase of 1,000 units/kg, 4,000 units/kg, and 8,000 units/kg. Plasma methionine concentration fell from pretreatment levels of ∼9 and 15 μmol/L to the limit of detection level by 30 minutes and remained at the limit of detection for 8 hours and <5 μmol/L for 12 hours in the monkeys receiving doses of 4,000 units/kg and 8,000 units/kg MEGC-PEG-rMETase. Therefore, 4,000 units/kg MEGC-PEG-rMETase was found to be sufficient to deplete plasma methionine for at least 8 hours (Table 1) ⇓ .

Pharmacokinetics of MEGC-PEG-rMETase.

Pharmacokinetics were determined after a single i.v. injection of MEGC-PEG-rMETase at doses of 2,000 and 4,000 units/kg. The plasma MEGC-PEG-rMETase concentration-time profiles for holoenzyme and apoenzyme are presented in Fig. 1 ⇓ . The calculated pharmacokinetic values are shown in Table 2 ⇓ . MEGC-PEG-rMETase showed a rapid reduction of plasma holoenzyme activity with a T_1/2 of 1.3 h. AUC and CLs (total body clearance) were 63.1 units/ml/hour and 63.4 ml/hour for 4,000 units/kg, respectively. For 2,000 units/kg, the holoenzyme T_1/2 was 1.7 hours with a AUC and CLs of 45.8 units/ml/hour and 43.6 ml/hour, respectively. A one-compartment model adequately described the disposition of the MEGC-PEG-rMETase holoenzyme activity. However, the plasma concentration-time curve of MEGC-PEG-rMETase apoenzyme activity was biexponential with an initial rapid distributive phase, followed by an elimination phase with an extended half-life of 90 hours for 4,000 units/kg and 143.3 hours for 2,000 units/kg. Apoenzyme AUC and CLs were 7,437 units/ml/hour and 0.54 ml/hr, respectively, for 4,000 units/kg, and 5,814 units/ml/hour and 0.34 ml/hour, respectively, for 2,000 units/kg. A two-compartment model consisting of a central and peripheral compartment was fitted to the plasma concentration-time profile of MEGC-PEG-rMETase plasma apoenzyme activity. The data suggests that PLP was rapidly lost from MEGC-PEG-rMETase after injection, which is consistent with observations made previously in mice (23) . The extended half-life of MEGC-PEG-rMETase apoenzyme activity indicates that PEGylation of rMETase has more effect on clearance of the apoenzyme than PLP stabilization in vivo.

Plasma MEGC-PEG-rMETase Activity in Monkeys Receiving Repeated Doses of rMETase.

In the monkeys treated with 4,000 units/kg MEGC-PEG-rMETase twice daily for 7 days, plasma MEGC-PEG-rMETase holoenzyme activity maintained the same concentration-time profile during the 7-day treatment and subsequent challenge period (Fig. 2A) ⇓ . The plasma apoenzyme MEGC-PEG-rMETase concentration-time profiles were increased on days 4 and 7 due to accumulation of the apoenzyme after repeated dosing (Fig. 2B) ⇓ .

Methionine Depletion in Monkeys Receiving Repeated Doses of MEGC-PEG-rMETase.

During the 7-day treatment period, plasma methionine in the MEGC-PEG-rMETase-treated monkeys was effectively depleted by 4,000 units/kg bolus twice daily i.v. Plasma methionine was maintained at <5 μmol/L for at least 12 hours after each dose of MEGC-PEG-rMETase. At 12 hours after each dose of MEGC-PEG-rMETase, the average plasma methionine levels on days 1, 4, and 7 were 3 μmol/L, 3.9 μmol/L, and 4.3 μmol/L, respectively (Fig. 3) ⇓ . During the following challenge period, the plasma average methionine levels at 12-hour time-points were slightly increased (Fig. 3) ⇓ . Because no neutralizing antibody was identified, the reason for this reduced methionine depletion efficiency needs to be additionally investigated.

Effect of MEGC-PEG-rMETase on Food Intake and Body Weight.

All of the monkeys treated with MEGC-PEG-rMETase or vehicle tolerated the 7-day treatment period. Reduced food intake and slight body weight loss occurred in both MEGC-PEG-rMETase-treated monkeys during the 7-day treatment period. Food consumption recovered after the end of the 7-day treatment period. The body weight loss in the two MEGC-PEG-rMETase-treated monkeys was 7% and 7.8% on day 7. Body weight gain occurred after the 7-day treatment ended.

Effect of MEGC-PEG-rMETase on Hematologic and Blood Chemical Parameters.

Hematology and blood chemistry examinations results are shown in Table 3 ⇓ . All of the hematology parameters were normal during the 7-day treatment except red blood cells and hemoglobin, which were found to decrease from day 3 of MEGC-PEG-rMETase treatment. Red blood cells and hemoglobin were reduced to 45% and 41% of pretreatment level, respectively, by the end of the 7-day treatment period indicating anemia. Blood chemistry determinations revealed no abnormalities in most liver function tests, renal function, glucose, lipids, and electrolytes in the MEGC-PEG-rMETase-treated monkeys compared with the controls and pretreatment values.

Immunologic Reactions to MEGC-PEG-rMETase.

No anaphylactic shock or other immunologic reactions occurred in the two monkeys given the three challenge injections at days 29, 50, and 71 of MEGC-PEG-rMETase after the 7-day repeated treatment.

Determination of Plasma Anti-MEGC-PEG-rMETase Antibodies.

Plasma-specific anti-MEGC-PEG-rMETase IgG antibodies were not detected until day 29 at which point a 10⁻² plasma antibody titer was detected in the MEGC-PEG-rMETase-treated monkeys. The challenge injections of MEGC-PEG-rMETase on days 29, 50, and 71 resulted in slightly increased plasma antibody titers up to 10⁻³ to 10⁻⁴ on day 71 (Table 4) ⇓ . The antibody levels elicited by MEGC-PEG-rMETase were much less than those elicited by naked rMETase seen in our previous monkey study (Table 5 ⇓ ; ref. 16 ). Plasma anti-MEGC-PEG-rMETase antibody was not identified as a neutralizing antibody, because no significant enzyme activity losses were observed when 2 units/mL MEGC-PEG-rMETase were incubated in vitro with anti-MEGC-PEG-rMETase antibody-positive serum. Further tests for neutralizing antibodies will be performed.

Histopathological Effects of MEGC-PEG-rMETase.

One monkey from the MEGC-PEG-rMETase-treated and one from the control group were sacrificed at the end of the study. There were no obvious gross lesions observed in the major organs of MEGC-PEG-rMETase-treated monkey at necropsy. The organ weights, including heart, liver, kidney, lung, spleen, brain, and adrenal glands were in the normal range. No gross histologic changes were observed in the tissues examined. Microscopic histopathological analysis will be carried out in detail.

Comparison of MEGC-PEG-rMETase and rMETase in Primates.

Two significant differences have been observed between MEGC-PEG-rMETase in the current report and naked rMETase in our previous report (16) . The most important is the lack of any immunologic reaction in the MEGC-PEG-rMETase-treated monkeys. In contrast, rMETase elicited anaphylactic shock in monkeys, unless they were pretreated with hydrocortisone. The second important result was the 90 to 143-hour half-life of MEGC-PEG-rMETase apoenzyme activity, compared with 2.5 hours for naked rMETase, indicating that the apoenzyme form of MEGC-PEG-rMETase persisted for very long periods in the monkey plasma (Table 5) ⇓ . As we have observed previously in mice (23) , PLP supplementation greatly extends the MEGC-PEG-rMETase holoenzyme activity in mice. Such studies will also be performed in primates to determine whether PLP supplementation can extend the half-life of holoenzyme activity of MEGC-PEG-rMETase.

In conclusion, the results of the present primate study present a profile with respect to half-life antigenicity and toxicity that suggest clinical potential of MEGC-PEG-rMETase.