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1 AntiCancer, Inc., San Diego, California;
2 Shionogi and Co., Ltd., Osaka, Japan;
3 NOF Corp., Tokyo, Japan; and
4 Department of Internal Medicine, University of Texas at Dallas, Southwestern Medical School, Dallas, Texas
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-deamino-
-mercaptomethane lyase (methioninase, METase) [EC 4.4.1.11] from Pseudomonas putida (1)
has been previously cloned and produced in Escherichia coli (2, 3, 4)
to target the methionine dependence of tumor cells. rMETase5
is found in Pseudomonas (Pp), Aeromonas, and Clostridium, but not in yeast, plants, or mammals (5)
. rMETase is a homotetrameric PLP enzyme of 172-kDa molecular mass. The biochemical reaction catalyzed by rMETase is shown below (Scheme 1).
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-family of PLP enzymes that catalyze ,-elimination and -replacement reactions, such as cystathionine -lyase, cystathionine -synthase, and O-acetylhomoserine o-acetylserine sulfhydrylase (6)
. In rMETase, tyronsine 114 has been shown to be important in -elimination of the substrate (6)
.
rMETase has been crystallized (5
, 7)
. The structure of rMETase has been determined at 1.7Å resolution using synchrotron radiation diffraction data and found to be a homotetramer with 222 symmetry. Two monomers associate to build the active dimer. The spatial fold of the subunits have three functionally distinct domains. Their quaternary arrangement is similar to those of L-cystathionine ß-lyase and L-cystathionine -synthase from E. coli (5)
.
Previous studies have extensively documented that a broad range of human tumors are sensitive to rMETase in vitro. The IC50 was several fold less for a wide variety of cancer cell lines compared to non-neoplastic cells. Sensitivity was particularly exquisite for breast, kidney, colon, lung, and prostate tumor cell lines (2 , 8) . Subsequent evaluation of rMETase on a variety of tumor cell lines in mouse xenograph models demonstrated a similar sensitivity to rMETase (8) . In addition, plasma methionine depletion by rMETase resulted in a remarkable increased sensitivity of the tumors to several different types of chemotherapeutic agents (9) .
However, the short in vivo half-life of rMETase and evidence of immunogenicity indicated the need to prolong the survival of the enzyme, prolong the period of methionine deprivation, and reduce potential immunogenicity that might result from repeated administration of the enzyme.
Conjugation of protein therapeutics with PEG has been shown to confer important therapeutic benefits—most importantly, increased serum half-life and reduced antigenicity (10) . PEG-proteins have enhanced solubility, decreased antigenicity, decreased proteolysis, and reduced rates of kidney clearance as well as enhanced selective tumor targeting. To couple PEG to a protein, it is first necessary to activate the polymer by converting the hydroxyl terminus to a functional group capable of reacting typically with lysine and N-terminal amino groups of proteins (10) .
Each ethylene oxide unit of PEG associates with two to three water molecules, which results in the molecule behaving as if it were five to ten times as large as a protein of comparable molecular weight (10) . The clearance rate of PEGylated proteins is inversely proportional to molecular weight (11) . Below a molecular weight of approximately 20,000, the molecule is cleared in the urine. Higher-molecular-weight PEG proteins are cleared more slowly in the urine and the feces (11) .
The FDA has approved the PEGylated forms of the protein therapeutics adenosine deaminase, asparaginase, -IFN and a growth hormone antagonist (12)
. PEG--IFN for treatment of hepatitis C (10
, 13) has recently been approved in two forms. Patients with refractory or recurrent acute lymphoblastic leukemia (ALL) are treated with a combination of PEG-asparaginase and methotrexate, vincristine, and prednisone (14)
. A genetic defect of adenosine deaminase (ADA) deficiency inhibits the development of the immune system making patients vulnerable to almost any type of infection. PEG-ADA strengthened the immune system considerably in these patients (15
, 16)
.
Initially, to prevent immunological reactions, which might be produced by multiple dosing of rMETase, and to prolong the serum half-life of rMETase, the N-hydroxysuccinimidyl ester of methoxypolyethylene glycol propionic acid (M-SPA-PEG 5000) was coupled to rMETase (17) . MALDI mass spectrometry indicated the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEGylation of rMETase increased the serum half-life of the enzyme in rats to approximately 160 min compared to 80 min for unmodified rMETase. PEG-rMETase could deplete serum methionine levels to less than 1 µM for approximately 8 h compared to 2 h for rMETase in rats (17) .
We report here the conjugation of methoxypolyethylene glycol succinimidyl glutarate PEG (MEGC-PEG) to rMETase with resulting remarkable prolongation of effective enzyme half-life and duration of methionine depletion in vivo. Simultaneous in vivo administration of pyridoxal 5'-phosphate along with MEGC-PEG-rMETase is shown to be a novel approach to improve the efficacy of the PEGylated enzyme.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Methoxypolyethylene glycol succinimidyl glutarate-5000 (MEGC-50HS-PEG or MEGC-PEG) (NOF Corporation, Kawasaki-shi, Kanagawa, Japan, Lot No. M21514) had a polydispersity of 1.02, substitution 94.2%, dimer content 0.84% and purity by 1H-NMR of 98.4%. The average molecular weight was 5461 Da. The chemical structure of MEGC-PEG is shown in Fig. 1
.
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Preparation and Purification of PEGylated rMETase.
The activated PEG derivative was used at a molar excess (1–4 fold) of PEG to free lysines in rMETase (32 per rMETase molecule), which corresponds to molar ratios of PEG to rMETase of 30–120/1. For each reaction, 120 mg/ml rMETase in 100 mM borate buffer (pH 8.8) was used. Based on 30–120/1 molar ratios of activated PEG versus rMETase (equal to 0.87–3.5/1 weight ratio of activated PEG versus rMETase), a given amount of the activated PEG was added to the rMETase solution with three stepwise additions at 30 min intervals. The PEGylation reactions were carried out at 20–25°C under gentle stirring for 90 min.
To eliminate an excess of unreacted activated PEG, the resulting PEG-rMETase conjugate was applied on a Sephacryl S-300 HR gel filtration column (HiPrep 26/60, Amersham Pharmacia Biotech, Piscataway, NJ, USA) immediately after the PEGylation reaction. PEG-rMETase was eluted with 80 mM sodium chloride in 10 mM sodium phosphate, pH 7.4, containing 10 µM PLP at a flow rate of 120 ml/h.
The fractions containing the PEG-rMETase conjugate were further purified by DEAE Sepharose FF column (XK 16/15, Amersham Pharmacia Biotech, Piscataway, NJ, USA) to remove trace amounts of un-PEGylated rMETase. The column was equilibrated and eluted with 80 mM sodium chloride in 10 mM sodium phosphate pH 7.2, containing 10 µM PLP at a flow rate of 180 ml/h. The fractions containing the PEG-rMETase conjugate were collected. PEG-rMETase-containing fractions were concentrated with an Amicon centriprep YM-30 (Millipore Corp, Bedford, MA, USA) and sterilized by filtration with a 0.22 µM membrane filter (Fisher Scientific, Tustin, CA, USA). Storage was at -80°C.
Determination of Protein Content.
Protein was measured with the Wako Protein Assay Kit (Wako Pure Chemical, Osaka, Japan) according to the instruction manual with slight modification (18)
. 50 µl of each sample or standard protein (BSA) was added to 3 ml of chromophore solution (pyrogallol red-molybdate complex) and vortexed well. The mixture was incubated at room temperature for 20 min without shaking and then measured for absorbance at 600 nm. The protein content of the sample was determined from the BSA standard calibration curve.
rMETase Activity Assay.
rMETase activity was determined from -ketobutyrate produced from L-methionine according to the method of Tanaka et al. (1)
with slight modification. 0.5 ml of sample diluted in 100 mM potassium phosphate buffer pH 8.0, containing 0.01% DTT, 1 mM EDTA Na2, 10 µM PLP and 0.05% Tween 80, was mixed with 0.5 ml substrate solution containing 100 mM potassium phosphate buffer, pH 8.0, 25 mM L-methionine and 10 µM PLP in a glass test tube. The reaction mixture was vortexed immediately and incubated at 37°C without shaking for precisely 10 min. The reaction was stopped by adding 0.5 ml of 50% TCA. The suspension was centrifuged at 13,000 rpm for 2 min. The supernatant (0.5 ml) was collected in a glass tube containing 1 ml 1 M acetate buffer, pH 5.0. Then, 0.4 ml MBTH solution containing 0.1% 3-methyl-2-benzothiazolinone hydrazone dydrochloride monohydrate (Wako Pure Chemical, Osaka, Japan) was added to the tube, mixed well and incubated at 50°C for 30 min. The absorbance of the reaction mixture was measured at 320 nm. The assay was carried out in triplicate. 
E 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 equation: Activity (U/ml) = 0.548 (1.07 + 2.2E) E. One unit of enzyme is defined as the amount of enzyme which produced 1 µM -ketobutyrate per minute at an infinite concentration of L-methionine.
SDS-Electrophoresis Analysis.
SDS-PAGE analysis of PEG-rMETase was carried out using 10% Novex polyacrylamide-precasted tris-glycine gels in Novex tris-glycine buffer with SDS according to the instruction manual. Gels were stained with Coomassie brilliant blue.
Determination of PEGylation Degree of rMETase.
The degree of modification of PEGylated rMETase was estimated both by the fluorescamine assay (19)
and by MALDI. For the fluorescamine assay, various amounts of rMETase and PEGylated rMETase in 2 ml of 0.1 M sodium phosphate buffer, pH 8.0 were mixed with 1 ml fluorescamine solution (0.3 mg/ml in acetone) and incubated for 5 min at room temperature. Samples were then assayed with a fluorescence spectrometer at 390 nm excitation and 475 nm emission. Results were plotted as fluorescence units versus concentration, with the slope of the line being determined by linear regression. The percent of PEGylated primary amines was determined according to the following formula: 1- (slope PEGylated rMETase/slope naked rMETase) x 100. MALDI analysis of naked and PEG-rMETase was performed at the Scripps Research Institute using a PerSeptive Biosystems Voyager-Elite mass spectrometer.
Plasma Methionine Determination.
The methionine level in the plasma was measured by pre-column derivatization, followed by HPLC separation (20)
. 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 min. 10 µl of the supernatant was mixed with 5 µl of a fluoraldehyde derivative reagent, o-phthaldialdehyde, for 1 min at room temperature, followed by addition of 150 µl of 0.1 M sodium acetate, pH 7.0. 20 µl of the reaction mixture was loaded on a reversed-phase Supelcosil LC-18DB column (25 cm x 4.8 cm, particle size 5 µm (Supelco, Bellefonte, PA, USA). The amino acid derivatives were separated by using a gradient elution of 40–60% solution B (Methanol) in solution A (tetrahydrofuran/methanol/0.1M sodium acetate. pH 7.2; 5/95/900) at a flow rate of 1.5 ml/minute. A fluorescence spectrophotometer was used for detection: 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.
Determination of Plasma PLP.
PLP in plasma was determined by HPLC using derivatization with sodium bisulfite in the mobile phase (21)
. Briefly, the plasma sample and PLP standard solutions were mixed with an equal volume of 0.8 M HCIO4 as deproteinizing agent, and vortexed vigorously. After centrifuging at 15000 rpm for 5 min at 4°C, the supernatants were taken and transferred to new vials. 50 µl of the supernatant was loaded on a reversed phase Cosmosil 5C18-AR-II column (4.6 x 150, Nacalai Tesque, Japan). The column was eluted with a gradient elution of 20–80% mobile phase B (30% acetonitrile/water v/v) in mobile phase A (0.1 M potassium dihydrogen phosphate buffer containing 0.1 M sodium perchloate and 0.5 g/L sodium bisulfite, pH 3.0) at a flow rate of 1.0 ml/minute. A fluorescence spectrophotometer was used for detection: excitation at 300 nm and emission at 400 nm. The PLP peak was identified by the retention time of a PLP standard. The concentration of plasma PLP was calculated using a calibration curve.
Pharmacokinetics and Methionine Depletion Efficacy in Vivo.
Athymic nude (nu/nu) mice aged 4 weeks (20–25g) were used for the study with 4 mice per group. Osmotic mini-pumps filled with 250 µl PLP (0.5g/ml) were implanted s.c. Twenty-four hours after pump implantation, 80 units native rMETase or PEG-rMETase in 0.5 ml PBS, pH 7.4, were injected via the tail vein in mice with or without PLP pumps. 400 µl blood was collected from the retrorbital plexus of each animal using heparinized capillary tubes. Blood was collected prior to injection, and 1 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h and 120 h post injection. The plasma was separated and stored in small aliquots at -80°C. The plasma enzyme activity, methionine concentration and PLP level in the plasma, collected at different time points, were measured as described above.
Determination of Plasma Anti-rMETase Antibody.
Normal BALB/c male mice were grouped randomly at 5 per group. Each mouse received three i.p. injections of 0.2 ml (200 µg) naked or PEG-rMETase emulsified in Freund’s complete adjuvant (FCA) at weekly intervals. Two weeks following the last injection, a booster injection of the rMETase or PEG-rMETase was given to each mouse. Blood samples were collected two weeks after the booster injection, and plasma was separated and stored at -80°C.
Plasma anti-rMETase antibody was measured using a Sandwich ELISA technique (22) . 100 µl 200 µg/ml rMETase in 0.1 M carbonate coating buffer, pH 9.5, was added to each well of a 96-well microplate and incubated at 4°C overnight. The plate was washed three times with PBS washing buffer, pH 7.4, containing 0.05% Tween-20, and blocked for 2 h at room temperature with 200 µl of 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 assay buffer were added to appropriate wells and incubated for 2 h at room temperature, followed by washing. 100 µl optimally diluted goat anti-mouse lgG and lgM subtypes conjugated with horseradish peroxidase (Sigma, St. Louis, MO) were added to each well. The plate was incubated for 1 h at room temperature and washed three times. 100 µl substrate solution containing O-phenylenediamine dihydrochloride (OPD) and hydrogen peroxide (Sigma) were added to each well, followed by 30 min incubation at room temperature. 50 µl 2N sulfuric acid were added to each well to stop the color reaction. The absorbance of each well was measured at 492 nm. The antibody titer was determined as the highest plasma dilution at which the extinction at 492 nm generated in the well of the immune plasma exceeded twice the extinction generated in those wells with negative control plasma.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. All rMETase subunits were PEGylated after 90 min reaction at room temperature when the above molar ratios were used since no non-PEGylated rMETase subunits were detected by SDS-PAGE (Fig. 2)
in the purified PEGylated rMETase conjugates. More PEG chains were conjugated to rMETase with increasing molar ratios of PEG to rMETase as seen by SDS-PAGE (Fig. 2)
. At low PEG/rMETase ratios, broad bands were observed on the gels, indicating the heterogeneity of PEGylated rMETase conjugates. When higher molar PEG/rMETase ratios were used in the reaction, less heterogeneity of PEGylated rMETase conjugates was observed (Fig. 2)
.
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. The fluorecamine assay indicated approximately 33%, 61% and 81% of free lysines in rMETase were coupled with PEG chains at ratios of PEG/rMETase-30/1, PEG/rMETase-60/1 and PEG/rMETase-120/1, respectively. The data indicated corresponds to an average of 3, 5 and 7 PEGylated lysines in each rMETase subunit, respectively, using the above coupling ratios.
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. These data are consistent with the covalent attachment of an average of 4, 6 and 8 PEG units to each rMETase subunit, respectively, for the three coupling ratios used. The signal peaks of PEGylated rMETase in the MALDI spectrum reflect the distribution heterogeneity arising from PEGylation of rMETase. It was found that at a higher PEGylation degree, fewer signal peaks in MALDI were observed, indicating less heterogeneity. For example, the PEG/rMETase 120 conjugate was the least heterogeneous (Fig. 3)
. Both MALDI and the fluorecamine assay demonstrate that an increase in PEG/rMETase molar ratios in the PEGylation reaction resulted in an increase in PEGylation of rMETase subunits.
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, Table 2A
).
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. However, PEG/rMETase-30, PEG/rMETase-60 and PEG/rMETase-120 depleted the plasma methionine level below 5 µM for 8, 24, and 48 h, respectively, which is 2-, 6-, and 12-fold longer than naked rMETase (Table 2B)
.
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. 80 U of naked or PEGylated rMETase were injected i.v. in animals with or without PLP supplementation from implanted mini-osmotic pumps. Blood samples were taken at different time points and measured for plasma methionine concentration. Plasma methionine depletion with PLP supplementation was prolonged significantly (Fig. 7)
compared to methionine depletion without PLP supplementation (Fig. 5)
, indicating PLP dependence for the in vivo methionine-depletion efficacy of PEG-rMETase. Plasma methionine levels depleted by the three PEGylated rMETase conjugates were maintained below 5 µM for 48 h. Plasma methionine levels were depleted below 5 µM for 72 h in case of PEG/rMETase 120 with PLP supplementation (Table 2B)
.
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versus that remaining after treatment with PEG-rMETase with PLP supplementation (PLP+) (Figs. 7
and 8
). These ratios showed that PEG/rMETase 60 and PEG/rMETase 120 had lesser PLP dependence than PEG/rMETase-30 (Figs. 5
, 7
, 8
), suggesting a protective effect of high-level PEGylation on retention of PLP by rMETase.
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, the plasma obtained from the mice immunized with PEG/rMETase-30, PEG/rMETase-60 and PEG/rMETase-120 produced IgG antibody titers of 10-7, 10-6, and 10-4, respectively, as compared to the titer of 10-8 produced from naked rMETase. For IgM antibody, an antibody titer of 10-3 was detected in the mice immunized with three PEGylated rMETase conjugates, which was lower than the antibody titer of 10-4 produced from naked rMETase.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effect of Extent of PEGylation on the Heterogeneity of the PEGylated rMETase Conjugate.
Heterogeneity of PEGylated rMETase was observed by both SDS-PAGE and MALDI. PEG, being a synthetic polymer, is polydispersed, which contributes to the heterogeneity of PEGylated conjugates. Ideally, a polydispersivity value (Mw/Mn) ranging approximately from 1.01 for low molecular weight oligomers (3–5 kDa), to 1.2 for high molecular weight (20 kDa) may be expected for PEGylation of proteins and peptides (23)
. Besides polydispersivity of PEG, it was found that the PEG/rMETase molar ratio influenced the heterogeneity of the resulting conjugate (Figs. 2
and 3
; Table 1
). Higher PEG/rMETase molar ratios resulted in less heterogeneity of the PEGylated rMETase conjugate.
Comparison of the Pharmacokinetics of MEGC-PEG-rMETase and M-SPA-PEG.
Pharmacokinetic data of the PEGylated rMETase conjugates indicated that the plasma circulating time depended on the PEGylation degree (Fig. 4)
. Compared to the plasma half-life of 160 min for the rMETase PEGylated with M-SPA-PEG (17)
, rMETase PEGylated with MEGC-PEG demonstrated a longer circulating time in blood. The half-life of PEG/rMETase-30, PEG/rMETase-60 and PEG/rMETase-120 was prolonged to 12 h, 18 h and 38 h, respectively (Table 2)
. This improved pharmacokinetic property may reflect the higher PEGylation efficiency of MEGC-PEG than compared to M-SPA-PEG for rMETase.
In Vivo Effect of PLP on PEG-rMETase.
We used 5 µM plasma methionine concentration as an end point for methionine depletion since it was reported that plasma methionine depletion below 5 µM was an effective therapeutic level of rMETase antitumor efficacy using mouse models of human cancer (24)
. Without PLP supplementation this level of methionine depletion could be achieved for 8 h by PEG/rMETase-30; 24 h by PEG/rMETase-60; and 48 h by PEG/rMETase-120. With supplementation, depletion to less than 5 µM methionine could be achieved for 48 h for PEG/rMETase-30 and PEG/rMETase-60; and 72 h by PEG/rMETase-120. These data suggest that PLP supplementation may be important for the antitumor efficacy of PEG-rMETase.
The protective effect of PEGylation on the apparent in vivo retention of PLP by rMETase was an unexpected result as was the rapid loss in vivo of PLP by naked rMETase and low-degree PEGylated rMETase. PLP in vitro is relatively tightly bound to rMETase (25) , but in vivo PLP appears to readily dissociate from rMETase. The recognition of the PLP effect which resulted in evidence of in vivo stabilization and retention of enzyme and prolonged efficacy in terms of reduction in plasma methionine levels is a most remarkable and potentially important therapeutic issue. The PLP effect further enhances the potential for utilization of PEG-rMETase as a clinical agent and suggests that it may have an important role in exploiting other PEGylated enzymes. The mechanism of dissociation of PLP from rMETase and apparent inhibition of dissociation by high-level PEGylation will be investigated in future studies.
Plasma anti-rMETase-specific antibody determination showed that PEGylated rMETase could reduce the antigenicity of rMETase in mice. Plasma IgG antibody is a critical antibody subtype which is related to hypersensitivity reactions and antibody neutralization of foreign proteins in vivo. PEG-rMETase demonstrated a significant decrease in antigenicity. For example, plasma anti-rMETase IgG antibody titer was reduced to 10-4 by PEG/rMETase 120 as compared to 10-8 for naked rMETase. Reduction in plasma IgG antibody depended on the number of PEG-derivatized amino groups, indicating the decreased antigenicity of PEGylated rMETase is a consequence of masking the protein antigenic sites by the polymer modification. Future studies will involve IgE studies, since this class of antibody may mediate hypersensitiviy/allergic reactions.
In conclusion, a high degree of modification of rMETase could be generated with MEGC-PEG with long-term retention of in vivo efficacy of methionine depletion. These PEGylated rMETase conjugates demonstrated increased pharmacokinetic efficacy by increasing plasma half-life and partially decreasing the PLP dependence of rMETase in vivo. Moreover, reduced antigenicity of rMETase was also achieved by PEGylated rMETase. Thus, molecular modification of PEGylation confers critical new properties to rMETase for its development as a novel cancer therapeutic.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Robert M. Hoffman, AntiCancer, Inc., 7917 Ostrow Street, San Diego, California 92111. Fax: (858) 268-4175; E-mail: all{at}anticancer.com
5 The abbreviations used are: rMETase, recombinant methioninase; PLP, pyridoxal 5'-phosphatase; MALDI, matrix-assisted laser desorption ionization; PEG, polyethylene glycol. 
Received 6/ 5/03. Revised 7/28/03. Accepted 8/ 1/03.
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REFERENCES |
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-deamino--mercaptomethane lyase (methioninase). Acta. Crystallagr. D Biol. Crystallogr., 56: 1665-1667, 2000.
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