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Recombinant methioninase (rMETase) was previously administered as an injectable drug to target methionine dependence of cancer. Recently, we observed that rMETase could be administered orally (o-rMETase) in a patient-derived orthotopic xenograft (PDOX) mouse model of melanoma. Here, we determined the efficacy of o-rMETase on a pancreatic cancer PDOX model. Forty pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each. o-rMETase was significantly more effective than i.p.-rMETase, but the combination of both was significantly more effective than either alone. Acquired gemcitabine resistance is a major factor in the recalcitrance of pancreatic cancer. We tested a human pancreatic cancer cell line, which has acquired >100-fold GEM-resistance (PK-9R) than its parental cell line PK-9. In contrast to GEM, both cell lines were very sensitive to rMETase. In orthotopic nude mouse models of PK-9 and PK-9R, GEM inhibited tumor growth in PK-9 but not PK-9R. In contrast, o-rMETase could inhibit both tumors. The combination of GEM + o-rMETase could regress the PK-9 tumor and inhibit PK-9R tumor growth. The present study shows that o-rMETase is effective and overcomes acquired GEM resistance in pancreatic cancer and demonstrates the clinical potential of this strategy.

Synovial sarcoma (SS) is a recalcitrant subgroup of soft tissue sarcoma (STS). A tumor from a patient with high grade SS from a lower extremity was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) mouse model. The PDOX mice were randomized into the following groups when tumor volume reached approximately 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks; G3, rMETase (100 unit/mouse, i.p., daily, for 2 weeks); G4 DOX (3mg/kg), i.p. weekly, for 2 weeks) combined with rMETase (100 unit/mouse, i.p., daily, for 2 weeks). On day 14 after treatment initiation, all therapies significantly inhibited tumor growth compared to untreated control, except DOX: (DOX: p = 0.48; rMETase: p < 0.005; DOX combined with rMETase < 0.0001). DOX combined with rMETase was significantly more effective than both DOX alone (p < 0.001) and rMETase alone (p < 0.05). The relative body weight on day 14 compared with day 0 did not significantly differ between any treatment group or untreated control. The results indicate that r-METase can overcome DOX-resistance in this recalcitrant disease.

We have previously established a patient-derived orthotopic xenograft (PDOX) model of undifferentiated spindle cell sarcoma (USCS). Recombinant methioninase (rMETase) has previously demonstrated efficacy in PDOX mouse models of human cancers. In the present study, we determined if rMETase in combination with doxorubicin (DOX) can overcome first-line DOX resistance in a PDOX models of USCS. The USCS PDOX mouse models were randomized into the following groups when tumor volume reached 100 mm3: G1, control without treatment; G2, doxorubicin (DOX) (3 mg/kg, intraperitoneal [i.p.] injection, weekly, for 2 weeks); G3, rMETase (100 units/mouse, i.p., daily, for 2 weeks); G4, DOX (3 mg/kg, i.p., weekly, for 2 weeks) combined with rMETase (100 units/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured twice a week. On day 14 after initiation, the USCS PDOX tumor sizes were (G1): 360 ± 85 mm3; DOX (G2): 355 ± 111 mm3, p = .927; rMETase (G3): 182 ± 57 mm3, p = .0003; DOX + rMETase (G4): 134 ± 29 mm3, p = .00001. These results indicate that rMETase can overcome USCS resistance to DOX, which is first line therapy for this disease. The body weight of treated mice was not significantly different in any group. The present results demonstrate the power of the PDOX model to identify effective therapy for recalcitrant cancer and the potential of rMETase to overcome DOX resistance.

Background: Ewing’s sarcoma (ES) is a recalcitrant disease in need of transformative therapeutics. Objectives: The aim of this study was to investigate the efficacy of tumor-selective Salmonella typhimurium A1-R combined with tumor metabolism targeting with oral administration of recombinant methioninase (o-rMETase), on an ES patient-derived orthotopic xenograft (PDOX) model. Methods: The ES PDOX models were previously established in the right chest wall. The ES PDOX models were randomized into 5 groups when the tumor volume reached 80 mm3: G1: untreated control; G2: doxorubicin; G3: S. typhimurium A1-R; G4: o-rMETase; G5: S. typhimurium A1-R combined with o-rMETase. All mice were sacrificed on day 15. Body weight and tumor volume were assessed twice a week. Results: S. typhimurium A1-R and o-rMETase respectively suppressed tumor growth as monotherapies (p = 0.050 and p = 0.032). S. typhimurium A1-R combined with o-rMETase regressed tumor growth significantly compared to untreated group on day 15 (p < 0.032). S. typhimurium A1-R combined with o-rMETase group was significantly more effective than S. typhimurium A1-R or o-rMETase monotherapy (p = 0.032, p = 0.032). Conclusions: The present results suggest that the combination of S. typhimurium A1-R and o-rMETase has promise to be a transformative therapy for ES.

Melanoma is a recalcitrant disease. Melanoma patients with the BRAF-V600E mutation have been treated with the drug vemurafenib (VEM) which targets this mutation. However, we previously showed that VEM is not very effective against a BRAF-V600E melanoma mutant in a patient-derived orthotopic xenograft (PDOX) model. In contrast, we demonstrated that recombinant methioninase (rMETase) which targets the general metabolic defect in cancer of methionine dependence, was effective against the BRAF-V600E mutant melanoma PDOX model. In the present study, we demonstrate that rMETase is effective against a BRAF-V600E-negative melanoma PDOX which we established. Forty BRAF-V600E-negative melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); temozolomide (TEM) (25 mg/kg, p.o., 14 consecutive days, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); TEM + rMETase (TEM: 25 mg/kg, p.o., rMETase: 100 units, i.p., 14 consecutive days, n = 10). All treatments inhibited tumor growth compared to untreated control (TEM: p = 0.0003, rMETase: p = 0.0006, TEM/rMETase: p = 0.0002) on day 14 after initiation. Combination therapy of TEM and rMETase was significantly more effective than either mono-therapy (TEM: p = 0.0113, rMETase: p = 0.0173). The present study shows that TEM combined with rMETase is effective for BRAF-V600E-negative melanoma PDOX similar to the BRAF-V600E-positive mutation melanoma. These results suggest rMETase in combination with first-line chemotherapy can be highly effective in both BRAF-V600E-negative as well as BRAF-V600E-positive melanoma and has clinical potential for this recalcitrant disease.

// Kei Kawaguchi 1, 2, 3 , Kentaro Igarashi 1, 2 , Shukuan Li 1 , Qinghong Han 1 , Yuying Tan 1 , Tasuku Kiyuna 1, 2 , Kentaro Miyake 1 , 2 , Takashi Murakami 1, 2 , Bartosz Chmielowski 4 , Scott D. Nelson 5 , Tara A. Russell 6 , Sarah M. Dry 5 , Yunfeng Li 5 , Michiaki Unno 3 , Fritz C. Eilber 6 and Robert M. Hoffman 1, 2 1 AntiCancer, Inc., San Diego, CA, USA 2 Department of Surgery, University of California, San Diego, CA, USA 3 Department of Surgery, Graduate School of Medicine, Tohoku University, Sendai, Japan 4 Division of Hematology-Oncology, University of California, Los Angeles, CA, USA 5 Department of Pathology, University of California, Los Angeles, CA, USA 6 Division of Surgical Oncology, University of California, Los Angeles, CA, USA Correspondence to: Robert M. Hoffman, email: all@anticancer.com Fritz C. Eilber, email: fceilber@mednet.ucla.edu Keywords: recombinant methioninase, methionine dependence, metabolic targeting, temozolomide, melanoma Received: June 06, 2017 Accepted: July 06, 2017 Published: August 12, 2017 ABSTRACT An excessive requirement for methionine termed methionine dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by methionine deprivation such as with recombinant methioninase (rMETase). The present study used a previously-established patient-derived orthotopic xenograft (PDOX) nude mouse model of BRAF V600E-mutant melanoma to determine the efficacy of rMETase in combination with a first-line melanoma drug, temozolomide (TEM). In the present study 40 melanoma PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n=10); TEM (25 mg/kg, oral 14 consecutive days, n=10); rMETase (100 units, intraperitoneal 14 consecutive days, n=10); combination TEM + rMETase (TEM: 25 mg/kg, oral rMETase: 100 units, intraperitoneal 14 consecutive days, n=10). All treatments inhibited tumor growth compared to untreated control (TEM: p =0.0081, rMETase: p =0.0037, TEM-rMETase: p =0.0024) on day 14 after initiation. However, the combination therapy of TEM and rMETase was significantly more efficacious than either mono-therapy (TEM: p =0.0051, rMETase: p =0.0051). The present study is the first demonstrating the efficacy of rMETase combination therapy in a PDOX model, suggesting potential clinical development, especially in recalcitrant cancers such as melanoma, where rMETase may enhance first-line therapy.

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.

An excessive requirement for methionine (MET), termed MET dependence, appears to be a general metabolic defect in cancer and has been shown to be a very effective therapeutic target. MET restriction (MR) has inhibited the growth of all major cancer types by selectively arresting cancer cells in the late-S/G2 phase, when they also become highly sensitive to cytotoxic agents. Recombinant methioninase (rMETase) has been developed to effect MR. The present review describes the efficacy of rMETase on patient-derived orthotopic xenograft (PDOX) models of recalcitrant cancer, including the surprising result that rMETase administrated orally can be highly effective.

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.

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 T

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/G

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.

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.

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.

Ewing's sarcoma (ES) is a recalcitrant disease in need of transformative therapeutics.The aim of this study was to investigate the efficacy of tumor-selective Salmonella typhimurium A1-R combined with tumor metabolism targeting with oral administration of recombinant methioninase (o-rMETase), on an ES patient-derived orthotopic xenograft (PDOX) model.The ES PDOX models were previously established in the right chest wall. The ES PDOX models were randomized into 5 groups when the tumor volume reached 80 mm3: G1: untreated control; G2: doxorubicin; G3: S. typhimurium A1-R; G4: o-rMETase; G5: S. typhimurium A1-R combined with o-rMETase. All mice were sacrificed on day 15. Body weight and tumor volume were assessed twice a week.S. typhimurium A1-R and o-rMETase respectively suppressed tumor growth as monotherapies (p = 0.050 and p = 0.032). S. typhimurium A1-R combined with o-rMETase regressed tumor growth significantly compared to untreated group on day 15 (p0.032). S. typhimurium A1-R combined with o-rMETase group was significantly more effective than S. typhimurium A1-R or o-rMETase monotherapy (p = 0.032, p = 0.032).The present results suggest that the combination of S. typhimurium A1-R and o-rMETase has promise to be a transformative therapy for ES.

Melanoma is a recalcitrant cancer. To improve and individualize treatment for this disease, we previously developed a patient-derived orthotopic xenograft (PDOX) model for melanoma. We previously reported the individual efficacy of tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) and recombinant methioninase (rMETase) for melanoma in the PDOX models of this disease. In the present study, we evaluated the efficacy of the combination of S. typhimurium A1-R with orally-administered rMETase (o-rMETase) for BRAF-V600E-negative melanoma in a PDOX model. Three weeks after implantation, 60 PDOX mouse models were randomized into six groups of 10 mice each: untreated control, temozolomide (TEM); o-rMETase; S. typhimurium A1-R; TEM + rMETase, S. typhimurium A1-R + rMETase. All treatments inhibited tumor growth compared to untreated control (TEM: p

In the present study, a patient-derived orthotopic xenograft (PDOX) model of recurrent cisplatinum (CDDP)-resistant metastatic osteosarcoma was treated with Salmonella typhimurium A1-R (S. typhimurium A1-R), which decoys chemoresistant quiescent cancer cells to cycle, and recombinant methioninase (rMETase), which selectively traps cancer cells in late S/G2, and chemotherapy. The PDOX models were randomized into the following groups 14days after implantation: G1, control without treatment; G2, CDDP (6mg/kg, intraperitoneal (i.p.) injection, weekly, for 2weeks); G3, rMETase (100unit/mouse, i.p., daily, for 2weeks). G4, S. typhimurium A1-R (5× 107 CFU/100μl, i.v., weekly, for 2weeks); G5, S. typhimurium A1-R (5× 107 CFU/100μl, i.v., weekly, for 2weeks) combined with rMETase (100unit/mouse, i.p., daily, for 2weeks); G6, S. typhimurium A1-R (5× 107 CFU/100μl, i.v., weekly, for 2weeks) combined with rMETase (100unit/mouse, i.p., daily, for 2weeks) and CDDP (6mg/kg, i.p. injection, weekly, for 2weeks). On day 14 after initiation, all treatments except CDDP alone, significantly inhibited tumor growth compared to untreated control: (CDDP: p = 0.586; rMETase: p = 0.002; S. typhimurium A1-R: p = 0.002; S. typhimurium A1-R combined with rMETase: p = 0.0004; rMETase combined with both S. typhimurium A1-R and CDDP: p = 0.0001). The decoy, trap and kill combination of S. typhimurium A1-R, rMETase and CDDP was the most effective of all therapies and was able to eradicate the metastatic osteosarcoma PDOX.

Pancreatic cancer is a recalcitrant disease. Gemcitabine (GEM) is the most widely-used first-line therapy for pancreatic cancer, but most patients eventually fail. Transformative therapy is necessary to significantly improve the outcome of pancreatic cancer patients. Tumors have an elevated requirement for methionine and are susceptible to methionine restriction. The present study used a patient-derived orthotopic xenograft (PDOX) nude mouse model of pancreatic cancer to determine the efficacy of recombinant methioninase (rMETase) to effect methionine restriction and thereby overcome GEM-resistance. A pancreatic cancer obtained from a patient was grown orthotopically in the pancreatic tail of nude mice to establish the PDOX model. Five weeks after implantation, 40 pancreatic cancer PDOX mouse models were randomized into four groups of 10 mice each: untreated control (n = 10); GEM (100 mg/kg, i.p., once a week for 5 weeks, n = 10); rMETase (100 units, i.p., 14 consecutive days, n = 10); GEM+rMETase (GEM: 100 mg/kg, i.p., once a week for 5 weeks, rMETase: 100 units, i.p., 14 consecutive days, n = 10). Although GEM partially inhibited PDOX tumor growth, combination therapy (GEM+rMETase) was significantly more effective than mono therapy (GEM: p = 0.0025, rMETase: p = 0.0010). The present study is the first demonstrating the efficacy of rMETase combination therapy in a pancreatic cancer PDOX model to overcome first-line therapy resistance in this recalcitrant disease.

Undifferentiated spindle-cell sarcoma (USCS) is a recalcitrant -cancer in need of individualized therapy. A high-grade USCS from a striated muscle of a patient was grown orthotopically in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. In a previous study, we evaluated the efficacy of standard first-line chemotherapy of doxorubicin (DOX), gemcitabine (GEM) combined with docetaxel (DOC), compared to pazopanib (PAZ), a multi-targeting tyrosine-kinase inhibitor, in an USCS PDOX model. In the present study, mice-bearing the USCS PDOX tumors were randomized into the following groups when tumor volume reached 100 mm3 : G1, untreated control without treatment; G2, DOX (3 mg/kg, intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, L-methionine α-deamino-γ-mercaptomethane lyase (recombinant methioninase [rMETase]) (100 U/mouse, i.p., daily, for 2 weeks). Tumor size and body weight were measured with calipers and a digital balance twice a week. The methionine level of supernatants derived from sonicated tumors was also measured. rMETase inhibited tumor growth, measured by tumor volume, compared to untreated controls and the DOX-treated group on day 14 after initiation of treatment: control (G1): 347.6 ± 88 mm3 ; DOX (G2): 329.5 ± 79 mm3 , P = 0.670; rMETase (G3): 162.6 ± 51 mm3 , P = 0.0003. The mouse body weight of the treated mice was not significantly different from the untreated controls. Tumor L-methionine levels were reduced after the rMETase-treatment compared to untreated control and pre-rMETase treatment. We previously reported efficacy of rMETase against Ewing's sarcoma and melanoma in a PDOX models. These studies suggest clinical development of rMETase, especially in recalcitrant cancers such as sarcoma.

The elevated methionine (MET) use by cancer cells is termed MET dependence and may be 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. We previously reported that rMETase, administrated by intra-peritoneal injection (ip-rMETase), could inhibit tumor growth in a patient-derived orthotopic xenograft (PDOX) model of a BRAF-V600E mutant melanoma. In the present study, we compared ip-rMETase and oral rMETase (o-rMETase) for efficacy on the melanoma PDOX. Melanoma PDOX nude mice were randomized into four groups of 5 mice each: untreated control; ip-rMETase (100 units, i.p., 14 consecutive days); o-rMETase (100 units, p.o., 14 consecutive days); o-rMETase+ip-rMETase (100 units, p.o.+100 units, i.p., 14 consecutive days). All treatments inhibited tumor growth on day 14 after treatment initiation, compared to untreated control (ip-rMETase, p

An excessive requirement for methionine (MET) for growth, termed MET dependence, appears to be a general metabolic defect in cancer. We have previously shown that cancer-cell growth can be selectively arrested by MET restriction such as with recombinant methioninase (rMETase). In the present study, we utilized patient-derived orthotopic xenograft (PDOX) nude mouse models with pancreatic cancer or melanoma to determine the relationship between intra-tumor MET level and tumor size. After the tumors grew to 100 mm3, the PDOX nude mice were divided into two groups: untreated control and treated with rMETase (100 units, i.p., 14 consecutive days). On day 14 from initiation of treatment, intra-tumor MET levels were measured and found to highly correlate with tumor volume, both in the pancreatic cancer PDOX (p

Aldesleukin is a recombinant form of IL-2 approved for metastatic melanoma and renal cell carcinoma that induced complete, durable remissions in certain patients. Yet, its use is infrequent because of vascular leak syndrome (VLS), a severe dose-limiting adverse event stemming from the engagement of the high affinity IL-2 receptor (IL-2R) alpha chain in type 2 innate lymphoid cells, eosinophils and vascular endothelial cells. THOR-707 is a site-directed, singly pegylated form of IL-2 completely lacking IL-2R alpha chain engagement yet retaining normal binding to the intermediate affinity IL-2R beta-gamma signaling complex expressed by natural killer (NK) and CD8+ T tumor-killing cells. We studied THOR-707 pharmacokinetics (PK) and pharmacodynamics (PD) in non-human primates (NHP) to evaluate peripheral biomarkers of immune cell activation (expansion of NK, CD8+ T and CD4+ regulatory T (Treg) cells and the induction of pSTAT5 and the proliferation marker Ki67). Here we show that in NHP, THOR-707 elicits the expansion of peripheral CD8+ T cells and the persistence of that response with different administration regimes. We also show that in the mouse syngeneic colon tumor model CT-26, THOR-707 induced Ki67 and the expansion of peripheral NK and CD8+ T cells. Within the tumor, THOR-707 promoted an increase in the numbers of infiltrating tumor-killing NK and CD8+ T cells without expansion of suppressive CD4+ Treg cells. Immunohistochemical (IHC) analysis showed that in THOR-707 treated animals, CD8+ T cells were mostly seen around the edges of the tumor and at areas of necrosis. Evaluation of T cell receptor (TCR) clonality revealed that treatment with THOR-707 increased intra-tumoral T cell diversity compared to the untreated animals. THOR-707 induction of CD8+ T cell tumor infiltration resulted in single agent dose-dependent anti-tumor efficacy, and additive efficacy in combination with PD-1 checkpoint inhibitor, increasing the survival of CT-26 tumor-bearing mice over either group alone. The pharmacodynamics, T cell infiltration and clonality, and efficacy of THOR-707 in two additional syngeneic models, have also been studied. Filing of an investigational new drug application for THOR-707 is expected in the second quarter of 2019 and thereafter initiation of a Phase 1/2 clinical trial in multiple tumor types as a single agent and in combination with an immune checkpoint inhibitor. Citation Format: Ingrid B. Joseph, Lina Ma, Jerod L. Ptacin, Carolina E. Caffaro, Hans R. Aerni, Kristine M. San Jose, Michael J. Pena, Robert W. Herman, Yelena Pavlova, David B. Chen, Ken Bragstad, Shukuan Li, Jasmine Nguyen, Laura K. Shawver, Lilia K. Koriazova, Marcos E. Milla. THOR-707, a novel not-alpha IL-2, elicits durable pharmacodynamic responses in non-human primates and efficacy as single agent and in combination with anti PD-1 in multiple syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3258.

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G0/G1 phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G2/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G0/G1 to S/G2/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G2, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G2 phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of "decoy, trap and shoot" chemotherapy.

Deprivation of methionine selectively arrests cancer cells during late S-phase (Proc. Natl. Acad. Sci. USA 77, 7306–7310, 1980; Biochim. Biophys. Acta, Reviews on Cancer 738, 49–87, 1984), where they are highly sensitive to chemotherapy drugs which damage DNA (J. Natl. Cancer Inst. 76, 629–639, 1986). Cancer cells, transformed to express different color fluorescent reporters during specific phases of the cell cycle (Cell 132, 487–498, 2008), were used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by recombinant methioninase (rMETase). The S/G2-phase blocked cancer cells fluoresced yellow or green in contrast to cancer cells in G1 which fluoresced red. Cancer cells, including MCF-7 breast cancer, synchronously blocked in S/G2-phase by rMETase, were identified by their yellow-green fluorescence and allowed to accumulate to the maximum extent. At the point of maximum yellow/green cells in the culture, the cells were administered chemotherapy drugs which interact with DNA or block DNA synthesis such as doxorubicin. We termed this procedure color-coded chemotherapy (CCC). CCC was highly effective against the cancer cells (90% cell kill). In contrast, treatment of cancer cells with drugs only, and without rMETase-effected S/G2-phase synchrony, led to the majority of the cancer cell population being blocked in G1 phase (red fluorescent) where they were resistant to the drugs (40% cell kill). CCC, which identifies, by fluorescent color, when cancer cells are blocked in S/G2-phase by a unique cell-cycle-blocking agent, rMETase, demonstrates the potential of cell-synchronization-based chemotherapy for breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-07-04.

Methionine depletion by recombinant methioninase (rMETase) has been demonstrated previously to be highly effective in tumor-bearing mouse models. However, the therapeutic potential of rMETase has been limited by its short plasma half-life and immunologic effects, including high antibody production in mice and monkeys and anaphylactic reactions in monkeys. To overcome these limits of rMETase, the enzyme has been coupled to methoxypolyethylene glycol succinimidyl glutarate (MEGC-PEG-5000). In this study, we evaluated the pharmacokinetics, antigenicity and toxicity of MEGC-PEG-rMETase in Macaca fascicularis monkeys using an escalating-dose strategy. Dose ranging studies at 1,000, 4,000, and 8,000 units/kg i.v. determined that a single dose of 4,000 units/kg was sufficient to reduce plasma methionine to

Recombinant methioninase (rMETase) has been shown to target the elevated methionine (MET) dependence of tumor cells and arrest their growth as well as make tumors more sensitive to standard chemotherapy agents. Polyethylene glycol (PEG)-modified rMETase (PEG-rMETase) has reduced antigenicity compared with unmodified rMETase. However, PEG-rMETase has a limited active circulating half-life due to rapid in vivo dissociation of its cofactor pyridoxal-5′-phosphate (PLP), a surprising finding, because PLP is tightly bound to PEG-rMETase in buffer. The question asked in the current study was on the effect of increasing doses of PLP to extend the circulating half-life of active PEG-rMETase holoenzyme in vivo. rMETase was 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. Pumps containing 500 mg/ml PLP increased the half-life of MEGC-PEG-rMETase holoenzyme 4.5-fold from 1.5 to 7 h. Infused PLP did not extend the half-life of MEGC-PEG-rMETase apoenzyme. With a dose of 4000 units/kg MEGC-PEG-rMETase in the mice infused with 5, 50, 200, and 500 mg/ml PLP, plasma MET was depleted from 50 μm to ≤5 μm for 8, 24, 72, and 72 h, respectively. Thus, PLP infusion could extend the period of MET depletion by MEGC-PEG-rMETase by ∼10-fold in a dose-dependent manner. The mice given 8000 units/kg MEGC-PEG-rMETase showed a similar plasma MET depletion time course, indicating that the limiting factor for MEGC-PEG-rMETase-mediated MET depletion in vivo was PLP. 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 infused PLP either bound to apo-MEGC-PEG-rMETase and/or inhibited dissociation of PLP from holo-PEG-rMETase, thereby maintaining the holoenzyme form of MEGC-PEG-rMETase in vivo. The combination of MEGC-PEG-rMETase treatment with PLP infusion suggests an effective clinical strategy for long-term MET depletion to arrest cancer growth.

Pharmacokinetics, methionine depletion, antigenicity, and toxicity of recombinant methioninase (rMETase), which has shown efficacy in achieving cell kill in a broad range of human tumor models, were examined in macaque monkeys. Dose-ranging studies at 1000, 2000, and 4000 units/kg i.v. identified the 4000 units/kg dose as able to reduce plasma methionine to an undetectable level (less than 0.5 μm) by 30 min, and the level so remained for 8 h. 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 methionine 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, which may be related to extensive blood sampling. Re-challenge on day 28 resulted in anaphylactic shock and death in one animal. Subsequent pretreatment with hydrocortisone prevented the anaphylactic reaction, although vomiting was frequently observed. Re-challenge was carried out at days 66, 86, and 116. Anti-rMETase antibodies (at 10−3) were found after the first challenge, and these increased to 10−6 after the fourth challenge and decreased to 10−2 by 2 months post therapy. The main rMETase antibody was IgG, and although it has some in vitro features of being a neutralizing antibody, each challenge dose was effective in depleting plasma methionine levels. Thus, rMETase was able to effectively deplete plasma methionine levels with minimal toxicity in a primate model. These data provide the bases for alteration by polyethyleneglycol conjugation (PEGylation) of the enzyme to increase its duration of effect and reduce its immunogenicity.

A major impediment to the response of tumors to chemotherapy is that the large majority of cancer cells within a tumor are quiescent in G0/G1, where cancer cells are resistant to chemotherapy. To attempt to solve this problem of quiescent cells in a tumor, cancer cells were treated with recombinant methioninase (rMETase), which selectively traps cancer cells in S/G2. The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination-based cell cycle indicator cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase blockage, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S/G2-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only, without rMETase-induced S/G2 phase blockage, led to the majority of the cancer-cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. In contrast, trapping of cancer cells in S/G2 phase by rMETase treatment followed by FUCCI-imaging-guided chemotherapy was highly effective in killing the cancer cells.

The methionine cycle and its metabolites homocysteine and cysteine serve several important functions in cellular metabolism. Abnormalities in metabolism of the methionine cycle have been associated with cancer. We determined plasma levels of methionine, homocysteine and cysteine in nude mice implanted with human cancer cell lines (MDA-MB-435 breast, PC-3 prostate, HT29 colon, BX-PC3 pancreas) over a prolonged period of tumor growth. The data were compared with correspondins values in nontumor-bearing controls. Nude mice were injected s.c. in the right flank with 10(6) cancer cells. Tumor growth was measured over time. Methionine was measured in plasma by HPLC. Cysteine and homocysteine were measured in plasma by recombinant enzyme assays and spectrophotometry to measure products. The concentrations of cysteine and homocysteine in plasma decreased significantly as a result of progression of breast, prostate and the pancreas tumor types implanted in the nude mice at least over a two-month period. Data for the colon tumors were nonsignificant for both cysteine and homocysteine. In the case of methionine, the decrease was significant only due to progression of the breast tumors, grown over a long time period, as compared to the mice without tumors control. The results suggest that sulphur amino acids may be plasma or serum biomarkers for cancer progression.

e13512 Background: A major impediment to the response of tumors to chemotherapy is that the large majority of cancer cells within a tumor are quiescent in G0/G1, where cancer cells are resistant to...

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) method for the separation and quantification of L-methionine in plasma has been developed. After derivatization of plasma amino acids with o-phthalaldehyde (OPA), a 50 microl sample was loaded on a reversed-phase Supelcosil LC-18-DB column (particle size 5 microm, 25 cm x 4.6 mm, 120A pores). A customized gradient program using tetrahydrofuran/methanol/0.1 M sodium acetate, pH 7.0, v/v/v=5/95/900 and methanol was used with detection by fluorescence. The elution time was 15 minutes, a 3-fold improvement over existing methods. The linearity was 1-100 microM. The limit of detection was 0.5 micromol/L, a 10-fold improvement over existing methods. The inter-assay CVs were 2-5%, and the intra-assays CVs were 4-8%. The sensitivity and rapidity of this HPLC method is particularly applicable to determine the efficacy of methionine depletion therapy of cancer.

Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo . The strain grows in tumor xenografts. In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice. S . typhimurium A1 is auxotrophic (Leu/Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally. Whether additional genetic lesions are present is not known. In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction. These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. In vivo , the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging. The bacteria, introduced i.v. or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice. By day 15, S . typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing. When the bacteria were injected intratumorally, the tumor completely regressed by day 20. There were no obvious adverse effects on the host when the bacteria were injected by either route. The S . typhimurium A1 strain grew throughout the tumor, including viable malignant tissue. This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy.

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.