Yuying Tan

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

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 <5 μmol/L for 12 hours. Pharmacokinetic analysis with the single 4,000 units/kg dose showed that MEGC-PEG-rMETase holoenzyme activity was eliminated with a biological half-life of 1.3 hours, and the MEGC-PEG-rMETase apoenzyme was eliminated with a biological half-life of 90 hours, an ∼36-fold increase compared with non-PEGylated rMETase. A single dose at 2,000 units/kg of MEGC-PEG-rMETase resulted in an apoenzyme half-life of 143 hours. A seven-day i.v. administration of 4,000 units/kg every 12 hours resulted in a steady-state depletion of plasma methionine to <5 μmol/L. The only manifest toxicity was decreased food intake and slight weight loss. Red cell values and hemoglobin declined transiently during treatment but recovered after cessation of treatment. Subsequent challenges on days 29, 50 and, 71 did not result in any immunologic reactions. This result is in contrast to non-PEGylated rMETase, which elicited anaphylactic reactions in monkeys. Anti-MEGC-PEG-rMETase antibodies (at 10−2) were found on day 29, and these increased to 10−3 to 104 on day 71, 100 to 1,000-fold less than antibodies elicited by naked rMETase. Although anti-MEGC-PEG-rMETase antibodies were produced, no neutralizing antibody was identified, and each challenge dose was effective in depleting plasma methionine levels. The results of the present study demonstrate that PEGylation greatly prolongs serum half-life of the rMETase apoenzyme and eliminated anaphylactic reactions. The results indicate a profile with respect to serum half-life, toxicity, and antigenicity that suggest clinical potential of MEGC-PEG-rMETase.

Metastatic patterns of lung cancer visualized live and in process by green fluorescence protein expression.

We demonstrate here the visualization of human lung cancer metastasis live and in process in nude mice by green fluorescent protein (GFP) expression. The human lung adenocarcinoma cell line Anip 973 stably transfected with the humanized GFP-S65T cDNA was selected for very bright green fluorescence. GFP-transfected lung cancer cells were initially inoculated subcutaneously in nude mice. Five weeks after transplantation, the resulting tumor had reached over 1 cm in diameter and had very bright GFP fluorescence. Fragments of subcutaneous tumor were implanted onto the visceral pleura of the left lung of nude mice by surgical orthotopic implantation (SOI) of histologically-intact tissue via transverse thoracotomy. The ipsilateral resulting tumor was highly fluorescent due to GFP expression. GFP expression allowed the visualization of the advancing margin of the ipsilateral tumor into the fresh normal lung tissue. Lymphogenous and direct-seeding metastases in the pulmonary hilum, cervical lymph nodes, the mediastinum and contralateral pleural cavity and contralateral lung in the SOI-treated mice were brightly visualized by GFP expression in fresh tissue. GFP-transfected and untransfected tumor had similar metastatic characteristics suggesting that GFP expression had no effect on metastasis itself. The results with the GFP-transfected tumor cells, combined with the use of SOI, demonstrate a fundamental advance in the visualization and study of lung cancer metastasis in process.

Pharmacokinetics, methionine depletion, and antigenicity of recombinant methioninase in primates.

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.

Circulating half-life of PEGylated recombinant methioninase holoenzyme is highly dose dependent on cofactor pyridoxal-5'-phosphate

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.

Survival efficacy of the combination of the methioninase gene and methioninase in a lung cancer orthotopic model.

We have previously demonstrated the antitumor efficacy of recombinant methioninase (rMETase) derived from Pseudomonas putida. To enhance the efficacy of rMETase, we have constructed the pLGFP-METSN retrovirus encoding the P. putida methioninase (MET) gene fused with the green fluorescent protein (GFP) gene. pLGFP-METSN or control vector pLGFPSN was introduced into the human lung cancer cell line H460. The methionine level of H460-GFP-MET cells was reduced to 33% of that of H460-GFP cells. rMETase (0.08 U/mL) in the medium resulted in 10% survival of H460-GFP-MET cells compared with untreated cells in vitro. In contrast, rMETase-treated H460-GFP cells survived at 90% of control. Tissue fragments harvested from subcutaneous tumors of H460-GFP-MET or H460-MET were implanted by surgical orthotopic implantation into the lungs of nude mice. A suboptimal dose of rMETase was administered intraperitoneally daily to mice in each group. Overall survival of rMETase-treated animals with H460-GFP-MET tumors was significantly longer than either rMETase-treated or -untreated animals with H460-GFP tumors (P < .05 in log-rank test). In two repeat experiments, rMETase-treated animals with H460-GFP-MET tumors had a 30-day survival of 80% and 83%, respectively. Untreated animals with H460-GFP-MET tumors had a 30-day survival of 40% and 58%, respectively. rMETase-treated animals with H460-GFP tumors had a 30-day survival of 0% and 33%, respectively. Untreated animals with H460-GFP tumors had a 30-day survival of 0% and 33%, respectively. The retrovirus-mediated gene transfer of METase decreased the intracellular methionine level of tumor cells and consequently enhanced the efficacy of treatment with the rMETase protein. We have thus demonstrated a new strategy of combination tumor therapy with the gene and gene product of MET.

Automated enzymatic assay for homocysteine

Various methods have been developed for plasma total homocysteine (tHCY) measurement, including a tHCY enzyme conversion immunoassay designed for the Abbott IMx analyzer (1), a microtiter plate tHCY enzymatic immunoassay assay (2), HPLC methods (3)(4)(5), and gas chromatography–mass spectrometry methods (6). We have described a single-enzyme tHCY assay (enzymatic tHCY assay) based on a highly specific recombinant form of l-homocysteine α,γ-lyase (rHCYase) (7)(8). We report here the application of this tHCY enzymatic assay on the Hitachi 912 automatic chemistry analyzer. The principle of the assay is that rHCYase produces H2S from tHCY and that the H2S is quantified by its reaction with N,N -dibutylphenylenediamine, which produces a chromophore. The assay uses four reagents and thus is compatible …

Combination treatment with recombinant methioninase enables temozolomide to arrest a BRAF V600E melanoma in a patient-derived orthotopic xenograft (PDOX) mouse model

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.

Cytotoxic synergism of methioninase in combination with 5-fluorouracil and folinic acid.

Potentiation of the cytotoxic activity of 5-fluorouracil (FUra) by folinic acid (5-HCO-H4folate) is due to elevation of the methylene tetrahydrofolate (CH2-H4folate) level, which increases the stability of the ternary complex of thymidylate synthase (TS), fluorodeoxyuridine monophosphate, and CH2-H4folate that inactivates the TS. Methionine deprivation results in the production of tetrahydrofolate (H4folate) and, subsequently, CH2-H4folate from methyl tetrahydrofolate, as a consequence of the induction of methionine synthesis. We hypothesized that the efficacy of FUra could be augmented by the combination of high-concentration 5-HCO-H4folate and recombinant methioninase (rMETase), a methionine-cleaving enzyme. Studies in vitro were performed with the cell line CCRF-CEM. Cytotoxic synergism of FUra + rMETase and FUra + 5-HCO-H4folate + rMETase was demonstrated with the combination index throughout a broad concentration range of FUra and rMETase. A subcytotoxic concentration of rMETase reduced the IC50 of FUra by a factor of 3.6, and by a factor of 7.5, in the absence and in the presence of 5-HCO-H4folate, respectively. 5-HCO-H4folate increased the intracellular concentrations of CH2-H4folate and H4folate from their baseline levels. Concentrations of folates were not changed by exposure to rMETase. Levels of free TS in cells treated with FUra + 5-HCO-H4folate and with FUra + rMETase were lower than those in cells exposed to FUra alone. The decrease of TS was still more pronounced in cells treated with FUra + 5-HCO-H4folate + rMETase. The synergism described in this study will be a basis for further exploration of combinations of fluoropyrimidines, folates, and rMETase.

Comparison of androgen-independent growth and androgen-dependent growth in BPH and cancer tissue from the same radical prostatectomies in sponge-gel matrix histoculture

In order to determine androgen sensitivities of prostate cancer and benign prostatic hypertrophy (BPH) tissues from the same patient in vitro, we used a histoculture technique to measure androgen‐independent and androgen‐dependent growth and compared them in paired specimens of BPH and prostate cancer from 23 radical prostatectomies. Both androgen‐independent growth and androgen‐dependent growth are measures of important biological characteristics of benign and malignant prostate tissue.

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

ABSTRACT 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.