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Methionine dependence is due to the overuse of methionine for aberrant transmethylation reactions in cancer. Methionine dependence may be the only general metabolic defect in cancer. In order to exploit methionine dependence for therapy, our laboratory previously cloned L-methionine α-deamino-γ-mercaptomethane lyase [EC 4.4.1.11]). The cloned methioninase, termed recombinant methioninase, or rMETase, has been tested in mouse models of human cancer cell lines. Ewing's sarcoma is recalcitrant disease even though development of multimodal therapy has improved patients'outcome. Here we report efficacy of rMETase against Ewing's sarcoma in a patient-derived orthotopic xenograft (PDOX) model. The Ewing's sarcoma was implanted in the right chest wall of nude mice to establish a PDOX model. Eight Ewing's sarcoma PDOX mice were randomized into untreated control group (n = 4) and rMETase treatment group (n = 4). rMETase (100 units) was injected intraperitoneally (i.p.) every 24 hours for 14 consecutive days. All mice were sacrificed on day-15, 24 hours after the last rMETase administration. rMETase effectively reduced tumor growth compared to untreated control. The methionine level both of plasma and supernatants derived from sonicated tumors was lower in the rMETase group. Body weight did not significantly differ at any time points between the 2 groups. The present study is the first demonstrating rMETase efficacy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as Ewing's sarcoma.

The tumor-specific increased minimal requirement for methionine has been shown to be a highly promising therapeutic target. To attack this target we have previously cloned the methioninase gene from Pseudomonas putida and produced recombinant methioninase (rMETase). A pilot Phase I clinical trial has been carried out to determine rMETase toxicity, rMETase pharmacokinetics, and serum MET-depletion in cancer patients. Patients with advanced breast cancer, lung cancer, renal cancer and lymphoma were given a single rMETase treatment at doses ranging from 5,000 to 20,000 units by i.v. infusion over 6-24 hours. No clinical toxicity was observed in any patient after rMETase treatment. rMETase levels reached 0.1 to 0.4 units per ml of serum in the patients which correspond to therapeutic levels in vitro. The lowest serum methionine levels in rMETase-treated patients were 0.1% of the pre-treatment levels corresponding to approximately 0.1 microM, which also correlates to therapeutic levels in vitro. The results of the rMETase pilot Phase I clinical trial therefore indicate that i.v. infusion of rMETase is safe and effectively depletes its biochemical target of serum methionine suggesting potential efficacy in future clinical trials.