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Methionine (MET) is a general metabolic therapeutic target in cancer, whereby cancer cells have an elevated requirement for MET, termed MET dependence. We have developed recombinant L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase, EC 4.4.1.11]) as targeted therapy of all cancer types. Pharmacokinetics, MET depletion, antigenicity, and toxicity of rMETase were examined in macaque monkeys. Pharmacokinetic analysis showed that rMETase was eliminated with a T1/2 of 2.49 h. A 2-week i.v. administration of 4000 units/kg every 8 h/day for 2 weeks resulted in a steady-state depletion of plasma MET to less than 2 μM. The only manifest toxicity was decreased food intake and slight weight loss. Serum albumin and red-cell values declined transiently during treatment. Rechallenge on day 28 resulted in anaphylactic shock and death in one animal. Pretreatment with hydrocortisone prevented the anaphylactic reaction. Anti-rMETase antibodies (at 10-3) were found after the first challenge, increased to 10-6 after the fourth challenge, and decreased to 10-2 by 2 months post-therapy. Therefore, the therapeutic potential of rMETase is limited by its short plasma half-life and immunologic effects, including high antibody production in mice and anaphylactic reactions in monkeys. To overcome these limits, rMETase has been coupled to methoxypolyethylene glycol succinimidyl glutarate polyethylene glycol (MEGC-PEG-5000). The pharmacokinetics, antigenicity, and toxicity of MEGC-PEG-rMETase in macaque monkeys were evaluated using an escalating-dose strategy. In pharmacokinetic studies, a single 4000 units/kg dose showed that MEGC-PEG-rMETase holoenzyme activity was eliminated with a biological half-life of 1.3 h, and the MEGC-PEG-rMETase apoenzyme was eliminated with a biological half-life of 90 h, a 36-fold increase compared with non-PEGylated rMETase. The disparity in the T½ of the apoenzyme and the holoenzyme reflects the loss of co-factor pyridoxal-L-phosphate of the circulating MEGC-PEG-rMETase. A 7-day i.v. administration of 4000 units/kg every 12 h resulted in a steady-state depletion of plasma MET to

Methionine (MET) has been shown to be a tumor-selective therapeutic target for cancer, since cancer cells require higher amounts of MET to divide and survive than normal cells. This phenomena is known as MET dependence and is probably due to MET overuse by cancer cells. A pilot clinical trial was initially carried out with non-recombinant METase (METase) produced from Pseudomonas putida and subsequently highly purified. No acute clinical toxicity was observed for any criteria measured in the three patients. The depletion of serum MET started within 30 min of the infusion and was maintained for 4 h after the infusion was completed in patient 1 and patient 2. The lowest serum MET levels were 35% and 19% of the pretreatment level, respectively, in patient 1 and patient 2. Patient 3 received a 10 h i.v. infusion of METase without any sign of side effects. MET was depleted over 200-fold from 23.1 to 0.1 μM by the 10-h infusion of patient 3. No clinical toxicity was observed in any criteria measured in patient 3. Subsequently, another pilot Phase I clinical trial was carried out of serum MET depletion in cancer patients by recombinant METase (rMETase) cloned from Pseudomonas putida and produced in E. coli. Patients with advanced breast cancer, lung cancer, renal cancer, and lymphoma were given a single rMETase treatment at doses ranging from 5000 to 20,000 units by i.v. infusion over 6-24 h. No clinical toxicity was observed in any patient after rMETase treatment. rMETase levels were measured at 0.1 to 0.4 units per ml of serum in the patients which correspond to therapeutic levels in vitro. The lowest serum MET levels in rMETase-treated patients were 0.1% of the pretreatment levels corresponding to approximately 0.1 μM, which also correlates to therapeutic levels in vitro as well as in vivo. The results of the METase and rMETase pilot Phase I clinical trials therefore indicate that i.v. infusion of rMETase is safe and effectively depletes its biochemical target of serum MET, suggesting potential efficacy in future clinical trials.

The elevated requirement for methionine (MET) of cancer cells is termed MET dependence. To selectively target the MET dependence of tumors for treatment on a large-scale preclinical and clinical basis, the L-methionine α-deamino-γ-mercaptomethane-lyase (EC 4.4.1.11) (methioninase, [METase]) gene from Pseudomonas putida has been cloned in Escherichia coli using the polymerase chain reaction (PCR). Purification using two DEAE Sepharose FF ion-exchange column and one ActiClean Etox endotoxin-affinity chromatography column has been established. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The recombinant bacteria produced rMETase at 43% of the total proteins in soluble fraction by simple batch fermentation using a 500 L fermentor. Crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100 L crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. Purified rMETase is stable to lyophilization. In order 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) has been coupled to rMETase. The PEGylated molecules (PEG-rMETase) were purified from unreacted PEG with Amicon 30 K centriprep concentrators or by Sephacryl S-300 HR gel-filtration chromatography. Unreacted rMETase was removed by DEAE Sepharose FF anion-exchange chromatography. The resulting PEG-rMETase subunit, produced from a PEG/rMETase ratio of 30/1 in the synthetic reaction, had a molecular mass of approximately 53 kda determined by matrix-assisted laser desorption/ionization mass spectrometry, indicating the conjugation of two PEG molecules per subunit of rMETase and eight per tetramer. PEG-rMETase molecules obtained from reacting ratios of PEG/rMETase of 30/1 had an enzyme activity of 70% of unmodified rMETase. 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 MET levels to less than 0.1 μM for approximately 8 h compared to 2 h for rMETase in rats. A significant prolongation of in vivo activity and effective MET depletion by the PEG-rMETase were achieved by the simultaneous administration of pyridoxal 5'-phosphate. rMETase was also conjugated with methoxypolyethylene glycol succinimidyl glutarate 5000 (MEGC-PEG). Miniosmotic pumps containing various concentrations of PLP were implanted in BALB-C mice. PLP-infused mice were then injected with a single dose of 4000 or 8000 units/kg PEG-rMETase. Mice infused with 5, 50, 100, 200, and 500 mg/mL PLP-containing miniosmotic pumps increased plasma PLP to 7, 24, 34, 60, and 95 μM, respectively, from the PLP baseline of 0.3 μM. PLP increased the half-life of MEGC-PEG-rMETase holoenzyme in a dose-dependent manner. The extended time of MET depletion by MEGC-PEG-rMETase was due to the maintenance of active MEGC-PEG-rMETase holoenzyme by infused PLP.

The elevated methionine (MET) requirement of cancer cells is termed MET dependence and is possibly the only known general metabolic defect in cancer. Targeting MET by recombinant methioninase (rMETase) can arrest the growth of cancer cells in vitro and in vivo due to their elevated requirement for MET. rMETase can also potentiate chemotherapy drugs active in S phase due to the selective arrest of cancer cells in S/G2 phase during MET restriction (MR). We previously reported that rMETase, administrated by intraperitoneal injection (ip-rMETase), could inhibit tumor growth in mouse models of cancer including patient-derived orthotopic xenograft (PDOX) mouse models. We subsequently compared ip-rMETase and oral rMETase (o-rMETase) on a melanoma PDOX mouse model. o-rMETase was significantly more effective than ip-rMETase to inhibit tumor growth without overt toxicity. The combination of o-rMETase+ip-rMETase was significantly more effective than either monotherapy and completely arrested tumor growth. Thus, o-rMETase is effective as an anticancer agent with the potential of clinical development for chronic cancer therapy as well as for cancer prevention. o-rMETase may also have potential as an antiaging agent for healthy people, since MR has been shown to extend the life span of a variety of different organisms.

Methionine (MET) is a general target in cancer due to the excess requirement of MET by cancer cells. MET has been effectively restricted by recombinant methioninase (rMETase) in mouse models of cell-line tumors. This chapter reviews the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) mouse models of human cancer. Ewing's sarcoma is a recalcitrant disease even though development of multimodal therapy has improved patients' outcome. A Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. rMETase effectively reduced tumor growth compared to the untreated control. The MET level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase treatment group. Body weight did not significantly differ at any time points between the two groups. A PDOX nude mouse model of a BRAF V600E-mutant melanoma was established in the chest wall of nude mice and also tested with rMETase in combination with a first-line melanoma drug, temozolomide (TEM). Combination therapy of TEM and rMETase was significantly more efficacious than either monotherapy. The results reviewed in this chapter demonstrate the clinical potential of rMETase.