TWI331531B - Pharmaceutical preparation and method of treatment of human malignancies with arginine deprivation - Google Patents

Description

1331531 BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition comprising arginase and use thereof. The present invention relates to a pharmaceutical composition which reduces arginine levels in a tumor patient and its use in the treatment of human malignancies. The invention also relates to a method of producing a recombinant protein.

[Previous technology j Background of the invention 10 arginase I (EC 3.5.3.1; L-arginine hydrazine hydrolase), is a kind

A key mammalian liver enzyme that catalyzes the final step of urea formation in the urea cycle's conversion of arginine to ornithine and urea. When a rat liver extract with high levels of arginase was accidentally added to tumor cell culture medium, it was found to have antitumor properties in vitro (Burton et al., 1967, corticosteroid 15 in vitro dissolution of thymus and lymphoma cells). Cellular action, Canadian biochemistry, 45, 289-297). Subsequent experiments showed that the anti-tumor effect of the enzyme is due to arginine depletion, which is an essential amino acid in the medium. When arginine is below 8 μΜ, cancer cells undergo irreversible cell death (Storr & Burton, 1974, Effect of arginine deficiency on lymphoma cells, Br. J. Cancer 30, 50-59 精. A newer aspect of the acid set in its role as a direct precursor of the strong signaling molecule nitric oxide (NO) synthesis, NO acts as a neurotransmitter, smooth muscle relaxant and vasodilator. The biosynthesis of NO involves Nitric oxide synthase (NOS) catalyzed Ca++ and NADPH-dependent reactions. Another recognized role of arginine 13351531 acid is its precursor to polyamines, spermidine and spermine, which participates in different Physiological processes 'including cell proliferation and growth (Wu & Morris, 1998, Arginine Metabolism: Nitric Oxide and Others, J. Biol. 336, 1-17). 5 Arginine is also an important enzyme such as nitric oxide synthase. Substrate for (NOS).

There are three types of NOS, nNOS, eNOS and iNOS, which convert the acid to nitric oxide and citrulline. Facial redness caused by NO is mediated, for example, by nN〇S, which is a neuronal type NOS. iNOS, an inducible NOS, is produced by macrophages, and NO produced in this way from arginine during septicemia causes vasodilation in endotoxic shock. eNOS, the endothelial NOS, is produced by endothelial cells in blood vessels, which converts arginine to NO and then causes platelet deaggregation on the surface of endothelial cells by the cGMP mechanism. The half-life of NO produced from eNOS in the local endothelial cell layer is about 5 seconds and the diffusion distance is about 2 microns. 15 The production of these enzymes is encoded by chromosomes 12, 17 and 7 respectively.

The NOS gene (NOS1 'NOS2, NOS3) is controlled. These genes present an amazingly similar genomic structure in terms of exon size and splice junction location. The in vitro anti-tumor effect of arginine depletion was recently confirmed by a research team in Scotland, UK (Scott et al., 2000, single amino acid (arginine) deprivation: transformation of cultured 20 and rapid and selective death of malignant cells, Br j

Cancer 83, 800-810; Wheatley et al., 2000, Single amino acid (arginine) restriction: growth and death of cultured HeLa and human diploid fibroblasts, Cell Physiology Biochemistry 10, 37_55). In all 24 different tumor cell lines tested, 'all cells died within 5 days of arginine, the cell line 6 1331531

K includes common cancer known as breast cancer's colorectal cancer, lung cancer, prostate cancer and ovarian cancer cell lines. Flow cytometry studies have shown that normal cell lines enter a resting state during the GO phase of the cell cycle up to several weeks without any significant injury. However, tumor cells enter the 5th phase of the G1 phase by the "R" point due to arginine deficiency. Arginine is an irreplaceable amino acid, and if there is no arginine, protein synthesis is disturbed... some cell lines show death from cell death. Even more exciting, repeated depletion can lead to tumor death without "resistance" (Lamb et al., 2000, single amino acid (arginine) deprivation causes Cdk4 phenotype in cultured human diploid fibroblasts Inhibition of (4) (1) stagnation 3, experimental cell research 225, 238-249). 15 20 Despite the promising data in vitro, it has not been successful to try arginine depletion in the body. Most of her (10) groups tried to treat the tumor-bearing rats with the liver in the abdominal cavity, but did not succeed (9) (10) & Bu_, 1974, the effect of arginine deficiency on lymphoma cells, 仏 c followed by 犷 & 50-59). At present, it is generally believed that under normal physiological conditions, blood (tetra) ammonia levels and other amino acid levels remain within the normal range _- _M) ‘muscle is the main hegemony. In the face of amino acid deficiency, the intracellular proteolytic pathway is broken (proteasome and lysosome), releasing amino acids into the circumstance _UmbfeS&Bafbadd, _, circulatory or non-circulating: key decisions in cancer, Nature Review 1, 22? ^, ., ± . A . _ 3l). The adaptive expansion of the amino acid species makes the various levels seem to be constant. Therefore, previous attempts to deplete arginine by multi-volume physical methods or arginine degrading enzymes have failed due to the amino acid adaptation mechanism of the organism. In order to overcome the problem of the natural adaptability of the body, the shirts and the like are described in U.S. Patent No. 6,261, < 557, the τ # ϋ therapeutic composition and a method for treating cancer, wherein a bismuth, citric acid is used. Decomposing enzymes are combined with a protein to inhibit the use of back-J, such as insulin, ur' acid. Preventing the body's muscles from replenishing depleted spermatorrhea Although insulin can be used as a s π with far-reaching physiology/decomposition inhibitors, its limited normality to the human body can be affected by the fact that the blood sugar level is not strictly maintained. problem. It is therefore an object of the present invention to find improved therapeutic methods and compositions for the treatment of cancer. C 明内 10 15 SUMMARY OF THE INVENTION Therefore, one product of the present invention

An isolated and substantially purified recombinant human arginase I (except for the Northern M F, otherwise referred to as arginine serotonin) is provided in a purity of 80-100. / . In a preferred embodiment, the arginase is of a purity. In a most preferred embodiment, the arginase of the present month is at least 99. /. pure. In the examples described below, the purine acid bismuth was determined to have a purity of 99.9% or more based on optical density after SDS_PAGE separation. In another preferred embodiment, the arginase enzyme of the present invention has a sufficiently high enzymatic activity and stability to maintain sufficient acid deprivation of at least 3 days in a patient" (adequate arginine) Deprivation, hereinafter referred to as AAD). A preferred modification is a 6-histidine amino terminal label. Another preferred modification is pegylation to increase the stability of the enzyme and minimize the immunoreactivity that occurs in the patient. In the examples described below, the arginase has a plasma half-life of at least about 3 days 8 1331531 and a specific activity of at least about 250 I.u./mg. In still another aspect of the invention, there is provided a method of producing a recombinant protein comprising (a) cloning a gene encoding the protein; (b) constructing a recombinant Bacillus subtiHs strain to express the protein (c) fermenting the recombinant B. subtilis cells using fed-batch fermentation; (d) heat-shocking the recombinant B. subtilis cells to stimulate expression of the recombinant protein' and (e) from said The recombinant protein is purified in the fermentation product. In a preferred embodiment, prophage is used as a recombinant strain. Cloning and expression of human recombinant arginase 10 using the above fed-batch fermentation method and prophage, increasing the maximum optical density (OD) at a wavelength of 600 nm by more than 4 times' and the arginase yield and yield Both were increased by more than 5 times as described in Example 3 below. In another embodiment, the fermentation step can be scaled up to produce recombinant protein. In yet another embodiment, the fermentation step is carried out using a known component fed-batch medium consisting of 18 〇 32 〇 15 glucosamine, 2-4 g/L mgs 〇 4 · 7 H 2 O, 45-80 g/L. The protein gland is composed of 7] 2 g/L IHPO4 and 3-6 g/L KH2P〇4. The use of a known component medium prevents the undesired material from being purified together with the recombinant protein, making the method safe and effective in the production of pharmaceutical grade recombinant materials. In another preferred embodiment, a human arginase gene having an additional coding region encoding six additional histidines at amino oxime* is provided, and the purification step comprises a chelating column chromatography step. In still another preferred embodiment, the arginase is further purified by pegylation to improve stability. In another aspect of the invention, there is further provided a pharmaceutical combination comprising an arginine 9 1331531 enzyme. In a preferred embodiment, the arginase has a sufficiently high enzymatic activity and stability to maintain at least 3 days of AAD in the patient. In a most preferred embodiment, the arginase is also modified by polyethylene glycolation to improve stability and minimize immunoreactivity. 5 According to another aspect of the invention, a pharmaceutical composition is further formulated using arginase. In another aspect of the invention, there is provided a method of treating a disease comprising administering to a patient a formulated pharmaceutical composition of the invention to maintain arginine levels in the patient below ΙΟμΜ for at least 3 days without the need for additional Egg 10 white matter decomposition inhibitor. In a preferred embodiment, insulin is not administered exogenously to a non-diabetic patient. In addition, the most preferred method of treatment of the present invention involves monitoring the patient's platelet count (preferably maintained at 50, above 109) and clotting time (not exceeding 2 times normal). The nitric oxide generator is not administered exogenously unless the above-mentioned 15 platelet count levels and clotting time are not reached.

In another preferred embodiment of this aspect of the invention, the pegylated arginase is administered in a short-term infusion over a period of 30 minutes at 3,000 to 5,000 I.U./kg. Arginine levels and arginase activity were measured daily before and after arginase infusion. If A AD is not reached the next day, the next infusion dose of the spermatoxin is determined according to the judgment of the treating physician. The maximum tolerated period of AAD is defined as the blood pressure is under control during this period (either by the treating physician or without drug control), the platelet count is above 50,000 x 109 and the clotting time is less than twice the normal value. Arginine levels were measured daily, whole blood count (CBC) and clotting time (PT). 肝 10 1331531 Liver function was monitored at least twice a week during treatment. The experimental data provided in the detailed description below shows that arginase can be used to treat malignant tumors if provided in a sufficiently strong form. Although recombinant human arginase I is a specific embodiment of the arginase used in the present invention, it should be clear that other forms of arginase and/or arginase from other sources may also be used according to the present invention. use. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows a plasmid map of PAB101. This plasmid carries the gene encoding arginase (arg) and is only replicated in E. coli, not in b. subtilis 10. Figure 2A, Figure 2B and Figure 2C show human arginase! The nucleotide sequence of the nucleotide and its deduced amino acid sequence. Figure 2A shows the nucleotide sequence of the EcoRI/MunI to Xbal site of plasmid pab 1 〇 1 (SEQ ID NO: 1). Column nucleotide (nt) 1 - 6, EcoRI/MunI site; nt 481 - 486, 15 -35 region of promoter ^; nt 504-509, promoter region -10 region; nt 544-549, promoter -35 region of 2; nt 566-571, region -10 of promoter 2; 600-605, ribosome binding site; nt 614-616, initiation of tambucus. nt 632 - 637 'Ndel site; nt 1601 — 1603 ' Stop codon; nt 199*7 — 2002, Xbal site. 2〇 Figure 2B shows the nucleotide sequence encoding the modified human arginase (SEQ ID NO: 2) and its corresponding encoded amino acid sequence (SEQ ID: 3). Nucleotides 616-1603 in Figure 2A are the coding regions for the modified arginase aminoguanidine sequence. The 6-histidine (SEQ ID NO: 4) label at the N-terminus is underlined. The translation stop codon is indicated by *. 11 1331531 Figure 2C shows the coding sequence of the normal human arginase I (SEq ID NO. 8) and its corresponding amino acid sequence (seq id NO: 9). Figure 3 is a schematic representation of the construction of B subtiHs prophage expressing arginase. 5 Figure 4A and Figure 5 show the fermentation time course of recombinant Bacillus subtilis strain LLC101 in a 2L fermentor. Figure 4A shows the results from batch fermentation. Figure 4B shows the results from the fed-batch fermentation. Figures 5A and 5B show the fermentation history' showing changes in parameters such as temperature, agitation speed, pH and dissolved oxygen values. Figure 5 shows a history of batch fermentation 10. Figure 5B shows a history of fed batch fermentation. Figures 6A and 6B show the results of biochemical purification of human arginase by the first 5 ml HiTrap column 3 hours after heat shock. Figure 6A shows FPLC operating parameters and protein elution profiles. Figure 6B shows the SDS_PAGE (12%) of the 5μ 1 11 - 31 fractions collected from the column. The protein gel was stained with Coomassie Brilliant Blue and destained to show the protein band. Lane Μ: low molecular weight range label (1 μ§/band; Bi〇 Rad), MW (Dalton): 97,400; 66,200; 45,000; 31,000; 21,500; 14,400. Figures 7A and 7B show the results of purification of human arginase by a second 5 ml 20 HiTraP binding column 3 hours after heat shock. Figure 7A shows FPLC operating parameters and protein elution profiles. Figure 7B shows an SDS-PAGE (12%) analysis of 1 μΐ 9 - 39 fractions collected from the column. Protein coagulation was stained with Coomassie Blue and destained to show protein bands. Lane Μ: low molecular weight range marker (1 pg/band; Bio-Rad), MW (dao_ton): 97,400 12 1331531; 66,200; 45,000; 31,000; 21,500; Figures 8A and 8B show the results of purification of human arginase by the first 5 ml HiTrap gel column 6 hours after heat shock. Figure 8A shows FPLC operating parameters and protein elution profiles. Figure 8B shows an SDS-PAGE (12%) analysis of 2·5 μl 10–32 fractions collected from the column 5 . The protein gel was stained with Coomassie Brilliant Blue and destained to show the protein band. Lane Μ: low molecular weight range marker (1 pg/band; Bio-Rad), MW (Dalton): 97,400; 66,200; 45,000; 31,000; 21,500; 14,400.

Figures 9A and 9B show the results of purification of human arginase by a second 5 ml 10 HiTrap coupling column 6 hours after heat shock. Figure 9A shows FPLC operating parameters and protein elution profiles. Figure 9B shows an SDS-PAGE (12%) analysis of 2 μΐ 8 -E6 fractions collected from the column. The protein gel was stained with Coomassie Brilliant Blue and destained to show the protein bands. Lane Μ: low molecular weight range marker (1 pg/band; Bio-Rad), MW (Dalton): 97,400 15; 66,200; 45,000; 31,000; 21,500; 14,400.

Figure 10 shows the time course of bacterial cell growth when heat shock is performed at a higher cell density. When the culture density (〇D6_m) was about 25, heat shock was performed at 8 hours. Figure 11 is a graph of the history of fed-batch fermentation when heat shock is performed at a higher density. This figure shows the parameter changes such as temperature, agitation speed, pH and dissolved oxygen values. Fig. 12A and Fig. 12B show the results of purification of human arginase by the first 5 ml HiTrap column after 6 hours of heat shock (higher cell density at OD25). Figure 12A shows the FPLC operating parameters and protein wash 13 1331531 off-line. Figure 12B. shows the results of SDS-PAGE (I2%) analysis of 5 μl of each fraction 16_45 collected from the column. The protein gel was stained with Coomassie Brilliant Blue and destained to show the protein bands. Lane Μ: marker of low molecular weight range (1 pg/band; Bi〇-Rad), MW (Dalton): 97,400; 66,200 5; 45,000; 31,000; 21,5〇〇; 14,400. Lane "crude extract,: 5μ1 crude extract of cells before loading in the column. Figures 13 and 13 show 6 hours after heat shock (higher cell density at 〇D25), pass the second Results of purification of human arginase by a 5 ml HiTrap column. Figure 13A shows FPLC operating parameters and protein wash 10 off. The figure shows 5 μΐ of each fraction collected from the column 7 - % SDS-PAGE (12 °/.) analysis results. Protein gel was stained with Coomassie brilliant blue and decolorized to show protein bands. Lane Μ: low molecular weight range label (1 pg/band; Bio -Rad), MW (Dalton): 97,400; 66,20〇; 45,000; 31,000; 21,500; 14,400. 15 Figures 14A and 14B show 6 hours after heat shock (higher cells at 〇D25) Density), the result of purification of human arginase by the first 1 ml HiTrapSP FF binding column. Figure 14A shows FPLC operating parameters and protein elution profiles. Figure 14B shows collection from the column 5 μιη of each fraction A11 — SDS-PAGE (12%) analysis of Β7. The protein gel was stained with 20 mas blue Color 'to show the protein band. Lane Μ: low molecular weight range of markers (1 pg / band; Bio-Rad), MW (to Dalton): 97, 4 〇〇; 66, 200; 45,000; 31,000; , 500 ; 14,400. Figures 15A and 15B show 6 hours after heat shock (higher cell density at 〇D25)' by humans]4L HiTrap SP FF binding column The results of the purification were performed. Figure 15A shows the FPLC operating parameters and the protein elution map. Figure 15B shows the SDS-PAGE (12 〇/〇) of 5 μΐ of each fraction Α6 - Β12 collected from the column. The results were analyzed. The protein gel was stained with Coomassie brilliant blue and destained to show the protein band. Lane Μ: low molecular 5 mile range marker (1 叩 / band; Bio-Rad) ' MW (Dalton) : 97,400 ; 66,200 ; 45,000 ; 31,000 ; 21,500 ; 14,400 ° Figure 16A and Figure 16B are SDS-PAGE (15%) of human arginase modified with mPEG-SPA (MW 5,0〇〇) For analysis, the ratio of arginase to PEG used was 1:50. Figure 16A shows the results when anti-10 on ice. Lane 1: low molecular weight range protein Lane 2: arginase without PEG (5.35 pg) (control group); lane 3: 1 hour after reaction; lane 4: 0.5 hour after reaction; lane 5: 2 hours after reaction; lane 6: reaction The last 3 hours; Lane 7: 4 hours after the reaction; Lane 8: 5 hours after the reaction; Lane 9: 23 hours after the reaction. Fig. 16B shows the results of the reaction at room temperature. Lane 1: low molecular weight range protein labeling; lane 2: arginase without PEG (5-35 pg) (control); lane 3: 1 hour after reaction; lane 4: 0.5 hour after reaction; lane 5: after reaction 2 Hours; Lane 6: 3 hours after the reaction; Lane 7: 4 hours after the reaction; Lane 8: 5 hours after the reaction; Lane 9: 23 hours after the reaction. 20 Figures 17A and 17B are SDS-PAGE (15%) analysis of human arginase modified with mPEG-SPA (MW 5,000) using a ratio of arginase to PEG of 1:20. Fig. 17A shows the result when the reaction was carried out on ice. Lane 1: low molecular weight range protein labeling; lane 2: arginase without PEG (5.35 pg) (control); lane 3: 1 15 1331531 after reaction

Hours; Lane 4.: 〇·5 hours after the reaction; Lane 5: 2 hours after the reaction, Lane 6: 3 hours after the reaction; Lane 7: 4 hours after the reaction; Lane 8: 5 hours after the reaction; Lane 9: After the reaction 23 hours. Fig. 17B shows the result of the reaction at room temperature. Lane 1: low molecular weight range protein labeling; lane 2: arginase without enzyme 5 (5.35 pg) (control group); lane 3: 1 hour after reaction; lane 4: 〇·5 hours after reaction; lane 5 2 hours after the reaction; Lane 6: 3 hours after the reaction; Lane 7: 4 hours after the reaction; Lane 8: 5 hours after the reaction; Lane 9: 23 hours after the reaction. Figure 18 is an SDS-PAGE (15%) analysis of human arginine 10 enzyme modified with mPEG-CC (MW 5,000). The reaction was carried out on ice. Lane 1: low molecular weight range protein labeling; lane 2: arginase without PEG (5.35 pg) (control); lane 3: 1:1 tyrosinase: 2 hours after PEG reaction; Lane 4: blank; Lane 5: arginase with 1:50 molar ratio: 23 hours after PEG reaction; Lane 6: arginase with 1:15 20 molar ratio: 2 hours after PEG reaction; Lane 7 : 1 : 20 molar ratio of arginase: 5 hours after PEG reaction; Lane 8 : with a 1: 20 molar ratio of arginase: 23 hours after PEG reaction. Figure 18B shows an SDS-PAGE (12%) analysis of natural arginase and PEGylated arginase, which are highly active and stable. Lane 1: low molecular weight range protein labeling (Bio-rad); lane 2: natural arginase (1 pg); lane 3: PEGylated arginase (1 μβ): lane 4: after ultradialysis Pegylated arginase (1.5 Hg). Figures 19A and 19B show the determination of the purity of the isolated recombinant human arginase. Figure 19A shows Lane 1 : 5 pg of purified E. coli expressed recombinant human arginase from Ikemoto et al., 16 1331531 (Ikemoto et al., J. Biol. Chem. 270, 697-703). Lane 2: 5 pg of purified recombinant human arginase expressed by B. subtilis from the method of the invention. Figure 19B illustrates the density analysis of the protein bands shown in Figure 19A using the Lumiianalyst 32 program of Lumi-imagerTM (Roche Molecular 5 Biochemicals). Upper: Results of Lane 1 of Figure 19A. Bottom: The result of lane 2 in Figure 19A.

Figure 20 is a graphical representation of the in vitro stability of PEGylated arginase in human plasma. 1 〇 21 and 22 show the half-life determination of the poly(ethylene glycol) arginase obtained in the method described in Example 8A in vivo. Figure 21 shows the in vivo activity of PEGylated arginase produced by the method of the present invention as measured using the activity assay described in Example 9A. Figure 2 2 is a graphical representation of the first half-life and the second half-life of the PEGylated arginase.

Figure 23 is a comparison of arginine depletion in four groups of laboratory rats administered intraperitoneally with different doses of PEGylated recombinant human arginase (500 1., 1000 LU., 150 IU And 3000 IU). Figure 24 shows the survival rate, mean tumor size, and tumor growth rate of the two groups of nude mice. The bare metal produced tumors by implanting Hep3B cells. One group was treated with a 5 〇〇ι_υ· dose of arginase in the glandular membrane, and the other control group was not treated with arginase. Figure 25 and Figure 25 show the mean tumor size and mean tumor weight comparison of two groups of nude mice that were injected with PLC/PRF/5 cells to produce 17 tumors & ~ intraperitoneal dose of 5 GG IU The arginase was treated in the same manner as the group without arginase treatment. Figure θ ί, Figure 26B shows the average tumor size of two groups of nude mice and the tumor weight of the nude mice. The nude mice produced tumors by implanting ΗιιΗ·7 cells. 5 °. The group secreted 5 〇Gl.U. The arginase was treated and the other control group was not treated with arginase. Figure 27 shows the average tumor size comparison of two groups of nude mice that were tumorigenic by implantation of MCF-7 cells. One group was treated intraperitoneally with 5 〇〇 π. β, and the other control group was not treated with arginase. 10 帛 2 8 and 2.9 show the arginine and CEA levels in the patient during the treatment as described in Example 12, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The term "pegylated arginase" as used in the present invention refers to the sperm (10) of the present invention modified by polyethylene glycol diolation to enhance the stability of the enzyme and Minimize immunoreactivity. The term "substantially the same as used in the present invention, whether it is a nucleotide sequence for DNA, a ribose acid sequence of RNA, or an amino acid sequence of a protein" means that the actual sequence revealed by Xiao Shuming has a small and non-phase and thus 20 Sequence of sequence changes. Species having substantially the same sequence are considered to be equivalent to the disclosed sequences and are included in the scope of the appended claims. In this respect, "small and non-phase-like sequence changes" means The sequences substantially identical to the DNA, RNA or protein disclosed herein and/or claimed herein are disclosed in the context of the invention, and the energy (IV). The euvalent sequence 18 1331531 functions in substantially the same manner, yielding a composition substantially identical to the nucleic acid and amino acid compositions disclosed and claimed herein. In particular, the DNA of the power phase b phase encodes the same protein as those disclosed herein, or encodes a protein with a conservative amino acid change, such as 5 replacing one non-polar residue with another non-polar residue, Alternatively, one charged residue is substituted for another similarly charged residue. These variations include those known to those skilled in the art that do not substantially alter the tertiary structure of the protein. The term "sufficiently high enzymatic activity" means that the recombinant human arginase has an enzyme specific activity of at least 250 IU/mg', preferably at least 300-350 IU/mg, more preferably at least 500 10 IU/mg. In a preferred In an embodiment, the arginase has a specific activity of 500 to 600 I.U_/mg. The term "stability" refers to the stability of the arginase in vitro. More preferably, the stability refers to stability in the body. The rate of decrease in enzyme activity is inversely proportional to the plasma stability of the isolated purified recombinant human arginase. The half-life of this human arginase in the blood system was calculated. 15 The term "sufficient arginine deprivation (AAD)" as used in the present invention means that the level of arginine in the body is 10 μΜ or less than 1 μ〇. The term "disease" refers to any pathological condition, including but not Limited to liver disease or cancer. The term "half-life" as used in the present invention means the time required for the concentration of the arginase to be reduced to half in human plasma in vitro. Earlier in the second year, the inventors were short of three cases in which hepatocellular carcinoma (HCC) spontaneously resolved spontaneously. All three patients spontaneously ruptured Hcc, leading to bloody peritonitis. In one case, plasma arginine levels were found to be as low as 3 μΜ and ascites in ascites at 7 μΜ. In these patients without any medication, their liver tumors spontaneously resolved after ruptured liver injury, while abortion 19 1331531 protein (AFP) normalized. One patient had his HCc subsided for more than 6 months. According to the present invention, it is believed that this prolonged regression is due to arginine depletion, which is caused by the spontaneous and sustained release of endogenous arginase from the ruptured liver to the peritoneum. Therefore, the inventors concluded that prolonged arginine depletion is a factor that causes HCC to resolve. 10 15 The inventors then designed a series of experiments showing that endogenous hepatic arginase can be released from the liver after transcatheter arterial embolization, resulting in systemic arginine deprivation. This has been filed in U.S. Provisional Patent Application No. 6/351,816. Xuan Shen π is incorporated herein by reference. In experiments designed by the present inventors, it was found that in patients with unresectable metastatic HCC, a moderately measurable amount was found after treatment with temporary hepatic perfusion _npiGd()1 and gelatinous hepatic artery embolization. (4) The liver arginine domain is placed in the systemic circulation. In the treatment program, a high-dose insulin infusion is added to produce a state in which the blood amino acid is too small. Of the 6 brain cases treated by f, 4 of the liver cancers of the liver were regressed, and the therapeutic effect was systemic. A liver and extrahepatic disease (abdominal adenosis) was continuously regressed by ρΕτ 20 radiation, and its AFP level fell to normal within 3 weeks and lasted for more than 4 months. CT scans at 4 months intervals showed no confirmed tumors in the liver and outside the liver. After 4 weeks of embolization, the other 3 patients had their extrahepatic disease disappeared. The lungs were at 2 j 疋 mesenteric / retroperitoneal / skeletal adenosis, and the latter was retroperitoneal adenopathy μ0,- All arginase activity and arginine levels were tested within 2 days'. All patients showed sufficient spermatozoa_蝎 duration to be closely related to the extent and duration of hepatic sputum 4 n in sputum and extrahepatic tumor regression. 0 20 1331531 Although transcatheter arterial embolization techniques are performed in conjunction with high-dose insulin infusion, the inventors have subsequently recognized in accordance with the present invention that the need to administer insulin is due to the fact that the arginase activity released into the patient's system is insufficient, Any protein degradation from the muscle has a compensatory effect on arginine deprivation and leads to ineffective treatment. In accordance with the present invention, the inventors have recognized that in order to improve the treatment and eliminate the need for insulin administration in combination with arginine deprivation therapy, a sufficiently high amount of serotonase activity must be present in the patient system to counteract any protein from the muscle. degradation. According to the present invention, the present inventors are therefore directed to producing an arginase having a sufficiently high activity and stability of enzyme 10 to maintain a sufficient arginine deprivation of less than 1 μM in a patient (hereinafter) Called AAD)" without the need to administer high doses of insulin. Thus, in addition to increasing endogenous arginase, the highly stable active arginase of the present invention provides the added benefit of achieving AAD without the administration of a protein degradation inhibitor that has undesirable side effects to the patient. Systemic depletion of arginine can cause other undesirable side effects associated with nitrogen oxide deficiency. These side effects include hypertension due to lack of NO to the vasodilatation of the endothelium, platelet aggregation and platelet reduction due to lack of NO and early clotting factor depletion associated with the temporary cessation of cell division. However, the inventors have verified that in the nitric oxide knockout mice, 20 animals have no hypertension and have a normal expected life of normal platelet count animals. Thus, according to another aspect of the invention and in patients with thrombocytopenia, no significant bleeding tendency is observed until the platelet count is below 50,000 χ 109. In patients with a tendency to bleed, the treatment must be such that the clotting time is extended to twice the normal rate. 21 The examples detailed below teach how to produce and use the sulphuric acid (IV) of the present invention. Example 1_ Construction of a recombinant strain of subtilis LLC1()1 containing human arginase gate recognition. ^ Group B.W hair _ two real calls in the recombinant LLC1 = heavy == test, in the L fermenter batch fermentation and supplement: t leaven. It was found that under batch conditions, a sufficiently high ^ could not be achieved. Only in this hair service & -_on. These experiments 10 Fermentation =:: Comparisons are shown in Example 3. Therefore, the fed batch was selected to produce the separated and purified heavy mash and the human extract was shortened. The ferment was scaled up to feed the fraction 1 core == Diwei was used in Example 2C. L money and results show that the LLC101 strain is a heat-sensitive strain that causes the arginase to swell at the machine. In the optimization test at the beginning of !7, the optimal conditions for producing the maximum amount of arginase in the heat of different cell densities. In Example 5, the cabinet describes the purification method and the pure feed obtained by two different fed batch fermentations of 12D and 12D (different density at 60 〇nm, density). Ammoxime production I ^ The experimental data shows that although all heat shocks are performed during the exponential growth phase of 20 LC101, heat shock at lower cell densities such as 0 12·800 produces better results. The maximum expression condition of arginase after heat shock is also optimized by changing the heat shock/text time. Example 4 shows the results of harvesting cells 3 hours after heat shock and using a fed-batch fermentation method. Example 5 illustrates the purification of the sperm 22 1331531 lysin 6 hours after the cell density of 12.8 OD heat shock. Example 6 illustrates the purification of arginase 6 hours after the cell density is 250D heat shock. Example 7 shows comparative data for arginase production under different harvesting and purification conditions. These data show that harvesting cells at 6 hours after a lower cell density heat shock of 12.8 produced a higher yield of arginase with a yield of 162 mg/L. The arginase is modified to improve stability. Example 8 A shows a method of PEGylated semen enzyme using cyanuric guanidine gas (cc) as a crosslinking agent' at a molar ratio of 1:140 (arginase: pEG). Example 8B illustrates a different pegylation process in which a lower proportion of crosslinker is added to the reaction mixture containing the enzyme. "Amber succinimide (SPA) with propionic acid was tested as a cross-linking agent. The experimental results show that the method using SPA described in Example SB provides a PEGylated synductase with a half-life of For 3 days, the specific activity was about 255, as discussed in Examples 9 and _. Example 8C illustrates a method for seeding (four) highly active PEGylated arginase, the specific activity of the arginase being about 592. 15 IU./mg 〇2〇

Using the above method, a highly stable (tetra) arginine function has been produced which has a sufficiently high (tetra) and silky therapeutic condition without the use of a white matter degradation inhibitor 'because any arginine supplemented by muscles Huiting 2 yolkase was quickly removed. Therefore, it is possible to achieve a arginine deprivation of less than 1 〇 _ without exogenous administration of insulin in large doses. Various treatments using Benzhi are described in Example u. The examples are similar to the clinical data of the arginase administered to the patient. ^ The therapeutic side h Μ step support embodiment. 13-14 is two animal experiments on rats, (4) 23 1331531 The dose response and safe dose of the acid enzyme. Examples 15-18 are another series of animal experiments performed on nude mice to investigate the response of tumors produced by different human cancer cell lines to arginine depletion produced by the administration of modified arginase. All references cited above are hereby incorporated by reference. The practice of the invention is illustrated in the following non-limiting examples. The scope of the invention is to be limited only by the scope of the appended claims. Example 10 Example 1: Construction of recombinant strain LLC101 (a) Isolation of the gene encoding human arginase, the gene sequence of human arginase I was published in 1987 (Haraguchi, Y. et al., 1987, Proc. 84, 412-415), based on the sequence designed primers. Polymerization 15 enzyme chain reaction (pCR) was performed using the Expand High Fidelity PCR System Kit (Roche) to isolate the gene encoding human arginase. Primers Argi (5^CCAAACCATATGAGCGCCAAGTCCAGAACCATA-S5) (SEQ ID NO: 5) and Arg2 (5'-CCAAACTCTAGAATCACA TTTTTTGAATGACATGGACAC-3') (SEQ ID NO: 6) were purchased from Singapore Genset Biotechnology Co., Ltd. These two primers have the same melting temperature (Tm) of 72 °C. Primer Argl contains a Ndel restriction enzyme recognition site (underlined). Primer Arg2 contains an Xbal site (underlined). The two primers (each with a final concentration of 300 nM) were added to a 5 μΐ human liver 5' stretch plus cDNA library (Clontech) in a 0.2 ml tube. In addition, DNA polymerase (2.6 units, 〇·75 μΐ), 4 deoxyribonucleotides (24 1331531 each)

The final concentration of each of 4 μb was 200 μM) and the reaction buffer (5 μl) and dH2O (17.75 μΐ). pcR was used for the following conditions: pre-PCR (94 ° C, 5 min), 25 PCR cycles (94 ° C, 1 min; 57 ° C, 1 min; 72 ° C '1 min) 'post-PCR (72 t:, 7 minutes). The PCR product (5 μM) was analyzed on a 0.8% 5 agarose gel to observe a 1.4 kb band. It is judged that this DNA contains a gene encoding arginase. (b) Isolated plasmid pSGll13 Plasmid pSGl 113 is a derivative of plasmid PSG703 (Thornewe U, SJ et al '1993, Gene, 133, 47-53), which was obtained by using Wizard Plus 10 Minipreps DNA Purification System (Promega) according to the instruction manual. Isolation was carried out from the E. coli DH5a clone carrying PSG1113. This plasmid was only replicated in E. coli and was not replicated in B. subtilis and used as a vector for subcloning the arginase gene. (c) subcloning the 1.4 kb PCR product into plasmid pSG1113 to form a plasmid pΑΒΙΟΙ. The PCR product prepared by the above method was subjected to restriction enzymes N de I and Xbal (Promega) in a reaction medium at 37°. C treatment for 1.5 hours, the medium consisted of 6 mM Tris-HCl (pH 7.9), 6 mM mgCl, 150 mM NaCl, 1 mM DTT. After the completion of the treatment, the reaction mixture was subjected to electrophoresis on a Joan 20-lipose gel (0.8%), and a 1.4 kb DNA fragment was recovered from the gel using a Qiaex II gel extraction kit (Qiagen). Plasmid pSG111 was treated in the same manner with the same restriction enzymes. After the completion of the treatment, the reaction mixture was subjected to agarose gel (0.8%) electrophoresis, and a DNA fragment of about 3.5 kb in size was recovered from the gel. The 25 1331531 DNA fragment was ligated to the above M kb DNA fragment using T4 DNA ligase. This ligation mixture was used to transform E. coli XLI-Blue using conventional calcium methods (Sambr〇〇k, J. et al., Molecular Cloning, A Guide to Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press, New York, 1989) On a nutrient agar 5 plate containing 1 〇〇pg/mi ampicillin. Colonies were screened and plasmids with appropriate inserts were analyzed by restriction analysis. This plasmid construct is called ρΑΒΙΟΙ (Fig. 1). 0RI is the origin of replication of E. coli, and bla is the ampicillin resistance marker gene. Primer Argi

(SEQ ID NO: 5), Arg2 (SEQ ID NO: 6) and Baqiao 6 (5, -CTCTGGCCATGCCAGGGTCCACCC-3,) (SEQ ID NO: 7) were sequenced into 10 rows of DNA to confirm the encoding of arginase Gene (Fig. 2). (d) Construction of a new recombinant B. subtilis prophage strain LLC101 Using the Wizard Plus Minipreps DNA Purification System (promega), plasmid pΑΒΙΟΙ was extracted from clones carrying ρΑΒΙΟΙ and purified. In the crude ρΑΒΙΟΙ (Fig. 1), the arginase gene (arg) is 〇.6 kb

15 Munl-Ndel φ105 phage DNA fragment (labeled "φ105") and cat gene (Fig. 1 and Fig. 3). This plasmid DNA (1 pg) was used to transform competent B. subtilis 1Α304 (φ105Μυ331) and transformed according to known methods (Anagnostopoulos C. and Spizizen J., 1961, Bacteriology 81, 741-746). The B. subtilis strain 1Α304 (φ105Μυ331) was obtained from J. 20 Errington (Thornewell, S. et al., 1993, Gene 133, 47-53). This strain was produced according to the method described in the following publication (Thornewell, S. et al., 1993, Genes 133, 47-53 and Baillie, LWJ et al., 1998, FEMS Microbiology Communication 163, 43-47), This is incorporated herein by reference in its entirety. The plasmid ρ ΑΒΙΟΙ (shown at the scribe in Fig. 3) was transformed into B. subtilis strain 1A304 ((j) 105 MU331) having a 26 1331531 selection marker (: 1 ), and the transformant was screened for the Ers phenotype. This transformant should have been generated from a double-crossover event. As shown in Figure 3, the transcription of the arginase gene (arg) is placed in a strong scorpion promoter (Leung and Erington, 1995, gene 154, 6) Under the control of 5, the thick line indicates the prophage genome, and the dotted line indicates B. subtilis chromosome 'thin line indicates plasmid DNA. The gene is shown in Figure 3. The black arrow indicates the direction of transcription and translation. The homologous region is marked with a vertical dotted line. , and the homologous recombination event was expressed by χ". 600 μΐ transformed cells were plated on agar 10 plate containing pneumomycin (5 μβ/ηιι), and 52 gasmycin resistance (CmR) was obtained therefrom. Colonies. 10 colonies were randomly selected and streaked on a plate containing erythromycin (2〇gg/mi). One of these colonies did not grow, indicating that it was erythromycin-sensitive (Ers). Isolating this pneumomycin-resistant but erythromycin-sensitive colony, Also named LLC101. In the chromosome of this newly constructed prophage strain, the red mold 15 gene resistance gene (ermC) was replaced by the arginase gene (arg) by double exchange in homologous recombination. 0.6 kb Munl-Ndel The φ105 sputum DNA fragment (labeled "φ105") and the cat gene provide homologous sequences for recombination. In this way, the arginase gene is directed to the original B. subtilis 1A304 ((M05MU331)) An expression site in phage DNA, and the sperm 2 prolinease gene is under the control of a strong temperature-inducible promoter (Leung, Y. C_ and Errington, J., 1995, genes 154, 1-6) B. Fermentation of SUBTILIS LLC101 cells Example 2A: Batch fermentation in a 2L fermenter B. subtilis LLC101 strain Wei. Holding nutrient agar supplemented with 5 mg/L chloramphenicol 27 < beef extract 1 g/L , protein gland 10 g / L, NaCl 5 g / L and Qiongqi Og / L) on the plate. To prepare inoculum for batch fermentation and fed-batch fermentation, several colonies of the above strains were prepared from freshly prepared nutrients Transfer the agar plates to two 1 L flasks, each containing ribs...fermentation medium, the medium Contains glucose 5g/L, trypsin gland 1〇g/L, yeast extract 3g/L, lemon 10 sodium citrate 1 g/L, KH2P04 丨.5 g/L, k2HP〇4 h5 g/L and (NH4 2S 〇 4 3 g/L. Bacterial cell cultures were cultured at 37 rpm c ^ DpH 7 in an orbital shaker at 250 rpm. The culture was stopped when the growth was about 9 - i i hours and the 〇 D6_m reached 5.5 - 6.0. Then, 16 〇_mL of the culture solution was introduced into a mash fermenter, which contained 144 〇 _mL fermentation medium (glucose 5 lbs, tensin protein gland 10 g / L, yeast paste 3 mp, ruby acid)纳机, ΚΑΡΟ, L5 g/L 'k2HP〇4 h5 g/L and (NH4)2S〇4 3 讥). Batch fermentation at °C. Controlled at 7 〇15 by the addition of sodium hydroxide and hydrochloric acid. The dissolved oxygen concentration is controlled to 20% air saturation by adjusting the (iv) speed. When the culture density (〇D_nm) is about 3 · At 9 o'clock, heat shock was performed at 3 25 hours. During the heat shock, the temperature of the fermenter was raised from 3rc to the thief, and immediately cooled to 3η: The heating and cooling cycle f was completed for 5 hours. After heat shock 3.5 Hours, the enthalpy of the culture reached a maximum of about (10). The cells were harvested 6 hours after 20 heat shock to separate and purify the sulphate.

The amount of active human arginase produced by the above strains was approximately gamma/L fermentation medium. The fermentation schedule is shown on the 4th touch. The changes in parameters, such as ^ degree, (iv) velocity, pH and dissolved oxygen value, are shown in Fig. 5A. Example 2B: Fed-batch fermentation in a 2 L fermentor 28 5 Feeding was carried out at 37 ° C, pH 7. 〇 and dissolved oxygen at 20% air saturation. The inoculation method was similar to that used in the batch fermentation described in Example 2A. The initial 'growth medium was used in the batch fermentation described in Example 2A. The soil is the same. The addition medium contained 200 g/L glucose, 2.5 g/L mgS〇4·7H2〇&apos; 50 team trypsin, 7.5 g/L K2HP04 and 3.75 g/L ΚΗ2Ϊ&gt;〇4. The media feed rate was controlled using a pH-stat control strategy. In this strategy, the feed rate was adjusted to compensate for the pH rise caused by glucose depletion. This fermentation strategy was first performed at about 4.5 hours of fermentation and when the glucose concentration dropped to a very low level. If pH &gt; 7.1, 4 mL of the flow-through medium is introduced into the fermenter. ^ After the addition of human glucose, the p-female immediately decreases rapidly below .1. After about 1 minute, when the added glucose was completely consumed by the bacteria, the pH was increased to 7 ux, indicating that another 4 ml of the addition medium should be added to the fermenter. # culture density Qingyi between (10) - 15 20 3.0 ' heat shock in 5-6 hours. During the heat shock, the fermenter's spoon 攸37C is picked up to 5〇〇c, and then immediately cooled to listen. It takes about 5 hours to complete the heating and cooling cycles. The blister is harvested in an hour after heat shock to separate and purify the arginase. Six hours after heat shock, the above strain produced a human arginase, in an amount of at least about the time course of fermentation of the brain. A graph showing the change in parameters such as temperature, lion speed, and yang = dissolution of the fed batch fermentation is shown in Figure 5B. Example 2C··Fed-batch fermentation in a 100 L fermentor The feed fraction was scaled up in a 100 L fermentor, and p was dissolved in (4) Cong air saturation. Initially, growth was established with a length of 10/〇. The medium used in the batch fermentation described in Example 2 was the same as that used in 29 1331531. The addition medium contained 3 g/L glucose, 3.75 g/L mg SCV 7H20, 75 g/L trypsin gland, h.25 g/L K2HP〇4 and 5.625 g/L KHzPO4. The media feed rate was controlled using a similar pH-stat control strategy as used in the batch fermentation described in Example 2B. At about 7.5 hours 5 heat shock was performed when the culture density (OD6 〇〇 mn) was between 11.5 and 12.5. During the heat shock, the temperature of the fermenter was increased from 37t to 5 (TC, at 5 (TC for 7 seconds, then immediately cooled to 37 ° C. It takes about 55 hours to complete the heating and cooling cycle. After heat shock 2 And 4 hours, cells were harvested to isolate and purify arginase. After 2 hours and 4 hours of heat shock, the above strains produced active human spermine 10 enzymes in amounts of at least about 74 mg and i24 mg/L fermentation medium, respectively. The ancient data show that in a 100 L fermentor, cells harvested 4 hours after heat shock produced higher yields of arginase than cells harvested 2 hours after heat shock. Example 3: Batch fermentation and supplementation Comparison of Batch Fermentation Table 1 compares the comparison between batch fermentation and fed-batch fermentation. Comparison of batch 15 fermentation, batch fermentation, batch fermentation, culture 〇D, arginase production spear: yield The aspect is even better.

20 Purified arginase, leaf Example 4. After low-cell density fed-batch fermentation, after the heat shock, the fermentation material was mixed as in Example 2 for fed-batch fermentation. The cell density of the fed-batch culture was monitored at 30 1331531 3〇 or 60-minute intervals. When the culture 0D reached 12.8 hours after the start of fermentation, the culture temperature was increased to capture heat shock (see Figures 4B and 5B). ). The cell culture collected 3 hours after heat shock (47 〇 at 4, it was centrifuged at 5 5,000 rPm for 20 minutes to pellet the cells. The net weight of the cells was i 5.丨g. Discard the culture and place the cells in the hall. Store at -80«&gt;c. At this temperature the cells can be stable for a few days. To extract intracellular proteins, resuspend the cell pellet in 14〇ml

Lotion buffer [50 mM Tris-HCl (pH 7.4), 0.1 M Nacn, 5 mM

MnS〇4 'lysozyme (75 pg/ml)]. After 15 minutes of incubation at 3 ° C, the 10 mixture was sonicated 8 times at 2 minute intervals using a Soniprep 150 instrument (MSE) for 10 seconds each (80 seconds total). About 5 units of deoxyribonuclease I (Sigma D 4527) was added, and the mixture was incubated at 37 ° C for 10 minutes to digest chromosomal DNA. After centrifugation at 10,000 rpm for 20 minutes at 4 ° C, the presence of arginase activity in the supernatant containing the crude protein extract was measured and analyzed by 15 SDS-PAGE (Laemmli, 1970, Nature 227, 680-685). A 5-ml HiTrap integration column (Pharmacia) was equilibrated with 5 column volumes of 0.1 MNiCl2 in dH20. A crude protein extract (140 ml) was loaded onto the column. 15 column volumes were eluted with a linear gradient (0-100%) at a flow rate of 5 ml/min under the following conditions: buffer A = initial buffer 2 mash [0.02 Μ sodium phosphate buffer (pH) 7_4), 0.5 MNaCl]; Buffer B = starting buffer containing 0.5 mM imidazole. The elution profile is shown in Figure 6A and the protein gel is shown in Figure 6. Fractions 13-20 (16 ml) were combined and diluted 10 times with the starting buffer [〇·〇2 Μphosphate buffer (pH 7.4), 0.5 M NaCl]. This was applied to a second 5-ml HiTrap chelating column (Pharmacia), 31 1331531. The same procedure as above was repeated. The elution is shown in Figure 7A and the protein gel is shown in Figure 7B. Fractions containing arginase 12-300 (38 ml) were combined, and a 50-ml HiPrep 26/10 desalting column (Pharmacia) was used to remove the salt under the following conditions: flow rate = 10 ml/min, buffer = 1 〇 mM Tris-HCl (pH 7.4) 5 , elution length = 1.5 column volume. The protein concentration was determined by the method described by Bradford (Bradford's Μ. Μ·' 1976, Anal. Biochem., 72, 248-254). A total of 56.32 mg of arginase was purified from 470 ml of cell culture. Purified arginase production was estimated to be n9.8 mg/l cell culture or 3.73 mg/g cell net weight. 10 Example 5: Purification of arginase 6 hours after heat shock after low cell density fed-batch fermentation As described in Example 4, fed-batch fermentation was carried out in a 2 L fermentor. The cell culture (650 ml) collected 6 hours after the OD 12.8 heat shock was centrifuged at 5,000 rpm for 20 minutes at 4 ° C to precipitate the cells. The net weight of the cells is 24g 15 . The culture supernatant was discarded and the cell pellet was stored at -80 °C. The cells will stabilize for a few days at this temperature. To extract intracellular proteins, the cell pellet was resuspended in 14 ml of lysis buffer [50 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 5 mM MnS〇4 'lysozyme (75 pg/ml)). At 3 〇. After 15 minutes of incubation, the mixture was sonicated 8 times at 2 minute intervals using a Soniprep 15(R) instrument (MSE) for 10 seconds (80 seconds total). Approximately 500 units of deoxyribonuclease I (Sigma D 4527) was added and the mixture was at 37. (: Incubation for 10 minutes to digest chromosomal DNA. After centrifugation at 10,000 rpm for 20 minutes at 4 ° C, the presence of arginase activity in the supernatant containing the crude protein extract was determined and passed through SDS-PAGE (Laemmli) , 1970 'Nature 227, 680-685) divided into 32. A 5-ml Η 鳌 鳌 ; ^ ^ ^ Pharmacia was equilibrated with 5 column volumes of 0.1 M NiCh in dH2 。. (14 〇 ml) was applied to the column. 15 column volumes were eluted with a linear gradient (0-100%) at a flow rate of 5 ml/min under the following conditions 5: buffer A = starting buffer [ 0.02 Sodium Phosphate Buffer (pH 7.4), 〇.5 M NaCl]; Buffer B = Starting Buffer containing 0.5 mM imidazole. The elution is shown in Figure 8 and the protein gel is shown in Figure 8. Combine fractions π - 24 (24 ml) and dilute 1 〇 〇 with starting buffer [0.02 Μ sodium phosphate buffer (pH 7_4), 0.5 M NaCl]. Add it to the second 5- The same procedure as above was repeated in a ml HiTrap integration column (Pharmacia). The elution is shown in Figure 9A and the protein gel is shown in Figure 9B. The fraction containing the benzoic acid enzyme 12 is combined. 24 (26 ml), using a 5 〇-ml HiPrep 26/10 desalting column (Pharmacia), remove the salt under the following conditions: Flow rate = 10 ml / min 'buffer = 1 〇 niM Tris-HCl (pH 7.4), 15 Elution length = 1.5 column volume. Protein concentration was determined by the method described by Bradford (Bradford, Μ·Μ· '1976, Analytical Biochemistry 72, 248-254). A total of 85.73 mg of arginine was purified from 650 ml of cell culture. Enzyme. Purified arginase production is estimated to be 132 mg/l cell culture or 3.57 mg/g cell net weight. 20 Example 6: Purification of arginase at 6 hours after heat shock at higher cell density in this particular feed In the batch fermentation, the procedure was similar to that described in the above examples except that the heat shock was carried out at 8 hours and the culture density (OD6G〇nm) was about 25. During the heat shock, the temperature of the fermenter was increased from 37 ° C to 50. °C, then immediately cooled to 37 ° C. This heating and cooling cycle is required to complete approximately 0.5 33 1331531 hours. A portion of the cell culture (760 ml) is harvested 6 hours after heat shock to isolate and purify the arginase. The time course of bacterial cell growth during fermentation is shown in Figure 1. The history of the fed-batch fermentation of temperature, mixing rate, pH and dissolved oxygen values is shown in Figure 11. 5 The cell culture (760 ml) collected 6 hours after heat shock was at 4 °C. Centrifuge at 5,000 rpm for 20 minutes to pellet the cells. The net weight of the cells was 32 g. Discard the culture supernatant and store the cells in -80μ. The cells are stable for a few days at this temperature. To extract intracellular proteins' resuspend the cell pellet in 280 ml to dissolve

Buffer [50 mM Tris-HCl (pH 7.4), 0.1 M NaQ, 5 mM 10 MnS〇4 'lysozyme (75 pg/ml)). After incubation for 15 minutes at 30 ° C, the mixture was sonicated 8 times at 2 minute intervals using a Soniprep 150 instrument (MSE) for 10 seconds each (80 seconds total). About 500 units of deoxyribonuclease I (Sigma D 4527) was added, and the mixture was incubated at 37 ° C for 10 minutes to digest chromosomal DNA. After centrifugation at 10,000 rpm for 20 minutes at 4 ° C, the presence of arginase activity in the supernatant containing the 15 protein crude extract was measured and analyzed by SDS-PAGE (Laemmli ' 1970, Nature 227, 680-685). A 5-ml Pharmacia was equilibrated with 5 column volumes of 0.1 M NiCh in dH2. A crude protein extract (280 mI) was loaded onto the column. 15 column volumes were eluted with a linear gradient (0-100%) at a flow rate of 5 ml/min under the following conditions 20: buffer A = starting buffer [0.02 Μ sodium phosphate buffer (pH 7 4) , 〇5 M NaC1]; Buffer B = starting buffer containing 0.5 mM imidazole. The elution profile is shown in Figure 12A and the protein gel is shown in Figure 12B. Combine the fractions 17 to 31 (3 〇, and use the starting buffer [〇·〇2 Μ Sodium Phosphate Buffer (pH 7 for fishing, 〇5 M Na(^) diluted 34 1331531 times. Add it to the second one. In the 5-ml HiTrap column (Pharmacia), the same procedure as above was repeated. The elution is shown in Figure 13A and the protein gel is shown in Figure 13B. The fraction containing arginase is 10-20 (22ml). Using a 50-ml·HiPrep 26/10 desalting column (Pharmacia), 5 salts were removed under the following conditions: flow rate = 10 ml/min, buffer = 10 mM Tris-HCl (pH 7.4), elution length = 1.5 Column volume. The sample was then loaded onto a 1-ml HiTrap SP FF column (Pharmacia) and eluted under the following conditions: flow rate = 1 ml/min, buffer A = 10 mM Tris-HCl (pH 7.4), Buffer B = 10 mM Tris-HCl (pH 7.4) with 1 M NaCl, linear gradient (〇 10 10 〇〇 ° / .) 'elution length = 30 column volumes. Elution diagram in Figure 14A, The protein gel is shown in Figure 14B. Fractions A12-B7 (7 ml) were combined and diluted 10-fold with starting buffer [0_02 Sodium Phosphate Buffer (pH 7.4), 0.5 M NaCl]. Two 1-ml HiTra In the p SP FF column (Pharmacia), the same procedure as above was repeated except that the fractional gradient was used for elution. The wash 15 is shown in Figure 15 and the protein gel is shown in Figure 15B. The combined fractions are 7-Β12. (7 ml)' and desalted using a 50-ml HiPrep 26/10 desalting column (Pharmacia). Protein concentration was determined by the method described by Bradford (Bradford, Μ·Μ., 1976, Analytical Biochemistry 72, 248-254). A total of 41.61 mg of arginase was purified from the 76 〇〇 11 cell culture. Purified arginase yield 20 was estimated to be cell culture or 1.3 mg/g cell net weight. Example 7: Harvesting and Purification under Different Conditions The arginase yield comparisons are shown in Table 2 below for the yields of arginine produced under different harvesting and purification conditions. These data are not produced in cells harvested at 128 hours after the lower cell density heat shock of 128. Higher yield of arginase, yield 35 after purification was 132 mg / L. Table 2 Fed-batch fermentation arginase yield (mg / L) 3 hours after heat shock harvest heat shock 6 hours after harvest OD 12. 8 heat shock 120 132 heat shock at OD 25 - 55.5 Example 8A: using cyanuric chloride (CC) Preparation of methoxy polyethylene glycol 5 t Enzymes 50mg of ethylene glycol was dissolved in arginase 2〇 ml PBS buffer solution (pH 7.4) to a final concentration of 2.5mg / ml. At 60. (: The arginase is heated for 1 min. After the start-up, the temperature of the enzyme is lowered to room temperature. The methoxypolyethylene glycol (mPEG-CC) activated by 丨g cyanuric urethane is used (MW= 5000, Sigma) was added to the arginase at a ratio of 1 • 140 molar (Arginase: PEG). The mixture was stirred using a magnetic stir bar until all the polyethylene glycol (pEG) was dissolved. When all PEG was dissolved, The PEG_arginase mixture &amp;pH was adjusted to 9.0 with 0.1 N NaOH, and the pH was further maintained at 9 Torr for 30 minutes by further addition of Na〇H. The poly 15 PEGylation was terminated by adding 01 n HCl to adjust the pH to 7.2. The PEGylated arginase was dialyzed against 2 3 3 liters of pbs buffer solution ρ Η 7·4 to remove excess PEG, and dialysis was performed using a Hemoflow F40S capillary dialyzer (Fresenius Medical Care, Germany). The PEGylated arginase was recovered and the final concentration was adjusted again. 20 The PEGylated arginase was filtered through a 0.2 μm filter into a sterile container and stored at 4 ° C. This enzyme is in the patient. The half-life of the body 36 1331531 has been tested for approximately 6 hours (see section 21)).

Example 8B: Preparation of arginase expressed in Β· SUBTILIS and use of CC or SPA 汆glycolation at low PEG ratios First by Davis, Abuchowski and colleagues (Davis, 5 F·F. et al., 978, Enzyme Eng. 4 '169-173) was revealed in the 1970s. In contrast to modifying pharmaceutical formulations, the chemical attachment of a poly(ethylene glycol) PEG component to a therapeutically useful protein (a known, PEGylated) represents a new approach that can enhance important drugs. Nature (Harris, J. M. Zun, 2001 'Clinical Pharmacokinetics, 539-551). 10 In 1979, S_ca et al. used 2,4,6-tris + triazine (cyanuric chloride) as a coupling. a 5,000 Dalton thiol polyethylene glycol (mpEG) 4 shells attached to bovine liver arginase (Sav〇ca, κ. V. et al., 1979,

Biochimica et Biophysica Acta 578 '47-53). The conjugate (PEG-arginase) retained only 65% of its original enzyme activity. They reported that 15 PEG_arginase has a longer life span than bovine arginase in the blood circulation of mice. The half-life of the injected bovine arginase is less than one hour. Their data also indicate that bovine arginase modified by P E G is non-immunogenic and non-antigenic when tested in mice. Recombinant human arginase (1. 〇 68 mg) was dissolved in 20 I25 borate buffer solution (pH 8.3) on ice or at room temperature. The mPEG to be activated, ie amber succinimide of mPEG propionic acid (mPE (3_SPA; MW 5 〇〇〇;

Shearwater C〇rporati〇n) or mPEG (mPEG-CC; MW 5,000; Sigma) initiated with cyanuric chloride, added to the solution at a molar ratio of 1:50 or 1:20 arginine I PEG. This is achieved by two steps 37 1331531. In the first step, half of the PEG is added to the arginase solution a little bit and gently mixed on ice for 30 minutes to prevent the pH from deviating from 8.0 to 8.5. range. The other half of the PEG was added to the solution and further gently mixed for 0.5 to 23 hours. This mixture was then dialyzed using a dialysis membrane with a cut-off value of less than 5 10,000, dialyzed against dH20 at 4 °C, and replaced at least 3 times with dH2. Both mPEG-SPA and mPEG-CC use the amino group of lysine and the N-terminus of the protein as a modification site. When arginase is modified at 1:50 arginase:PEG molar ratio on ice or at room temperature with 1 : £0-8? eight (1\^¥ 5,000), after 1 hour of reaction 10 Most enzyme molecules are modified (Figure 16). The sample showed the same state even after 23 hours of reaction. Immobilization of arginase molecules with different amounts of PEG molecules produces molecules of different molecular weights. As expected, a higher ratio of unpegylated forms of arginase was found when PEGylation was carried out using a lower molar ratio of 1:20 (Section 17). However, for the two arginase enzymes used in 15: PEG molar ratio, longer reaction times and the use of room temperature rather than on ice did not seem to affect the degree of PEGylation. In the case of mPEG-SPA (MW 5,000), a molar ratio of 1:50 and a reaction time of 1 hour, the arginase retains 72-76% of its original activity (see Table 3). Reported bovine arginase 20 activity (65%; Savoca, KV et al, 1979, BiochimiCa et Biophysica Acta 578, 47-53). When arginase was modified with mPEG-CC (MW 5,000) at 1:50 arginase:PEG molar ratio on ice, the reaction was very slow and it took 23 hours to complete PEGylation (Figure 18A) . In addition, most of the enzyme molecules 38 transform into a narrow range of very high molecular weights. If a lower molar ratio of 丨:20 is used, the reaction will be slower, as shown in Figure 18A. Example 8C: Preparation of highly active PEGylated arginase A fed-batch fermentation was carried out as described in Example 4 in a 15 L B. Braun Biostat C stainless copper 5 fermentor. The cell culture (8_4 L) collected at 4.6 hours after the heat shock of OD 12-13 was centrifuged at 5 °C for 2 minutes at 4 °C to precipitate the cells. Discard the culture supernatant and store the cell pellet in _8. The cells were allowed to stabilize at this temperature for several days. To extract intracellular proteins, the cells were resuspended in 1250 ml of lysis buffer [5 〇 Tris 'Hcl (pH 7.4), 10 〇.1MNaC1 '5 mM MnS〇4, lysozyme (75 pg/ml)). At 30. After incubating for 20 minutes, the mixture was divided into several beakers at a rate of 3 〇〇 n^, and each of the Soniprep 150 instruments (MSE) was ultrasonically spun 12 times at 2 minute intervals for 10 seconds each time ( A total of 12 ^ seconds ^ Add about 5 〇〇〇 unit deoxyribonuclease I (Sigma D 4527), and the mixture was incubated at 37t: 1 minute, 15 to digest chromosomal DNA. Centrifuge at 9,000 rpm at 4 ° C 30 After a minute, the presence of arginase activity in the supernatant containing the crude protein extract was determined and analyzed by SDS-PAGE (Laemmli '197〇, Nature 227, 68〇_685). Filtration of crude protein extract (1195) Ml) is divided into two parts, each containing 597.5 ml. Each part is then loaded onto the i3〇_mi Ni_NTA Qiagen column (Pharmada) using a linear gradient (〇-1%) The 5 ml/min flow rate was eluted under the following conditions: buffer A = starting buffer [0.02 Μ sodium phosphate buffer (pH 7.4), 0·5 M NaCl]; buffer B = containing 0.5 M imidazole Start buffer. Combine fractions containing pure arginase and replace the buffer with PBS buffer pH 7.4 at 35 ml/min at 4 °C. 9 1331531

Pellicon XL equipment (polyether-3⁄4 membrane cutoff = 8 kDa) and laboratory scale tangential flow filtration system (Millipore) for this operation. Protein concentration was determined by the method described by Bradford (Bradford, Μ. M., 1976, Analytical Biochemistry 72, 248-254). A total of 788 mg of arginase was purified from 8.4 L of cell culture. Purified arginase production was estimated to be 94 mg/l cell culture. The specific activity measured was as high as 518 I.U./mg. High specific activity PEGylated arginase was prepared in PBS buffer. Purified arginase (specific activity = 518 I.U./mg) was placed in PBS buffer prior to PEGylation. mPEG-SPA, MW 5,000 10 (5.82 g) was slowly added to a 555 ml solution of purified arginase (813.64 mg, 1.466 mg/ml) in a 1 L beaker, then stirred at room temperature for 2 hours and 40 minutes (fine Lysinase: molar ratio of mPEG-SPA = 1: 50). This mixture was then thoroughly dialyzed by ultrafiltration using 15 ml of PBS buffer using an F50(S) capillary dialyzer (Fresenius Medical Care) to remove all PEG that was not incorporated. mPEG-SPA uses the amino group of lysine and the N-terminus of the protein as a modification site. The specific activity of the PEGylated arginase was determined to be as high as 592 I.U./mg. The results of SDS-PAGE analysis of natural arginase and pegylated arginase are shown in Figure 18B. The PEGylated arginase was shown to be highly stable in terms of arginine 2 酶 enzyme activity and protein concentration when stored in the img/m^pBS buffer for at least 3 weeks at room temperature. When stored at 4 mg/ml in PBS buffer at 4 〇c, it was stable for at least 6 months without lowering the specific activity. 40 1331531 Table 3: Time (hours) of arginase activity (%) when the arginase activity was ligated with various activated PEG at different molar ratios and temperatures. Warming 1:20 arginase: mPEG-SP A ratio for PEGylation) Arginase activity (%) (Polymerization on ice with 1:20 arginine: mPEG-SP A ratio) Glycolation) Arginase activity (%) (Pegylated at room temperature with 1 • 50 arginase: mPEG-SP A ratio) Arginase activity (%) (Used on ice 1:50 arginase: mPEG-SP A ratio for PEGylation) Arginase activity (%) (for use on ice): 20 arginase: mPEG-C C Erythrocyte enzymatic activity (%) (on a 1:50 arginase: inPEG-C C ratio on ice. Polyethylene 2 100 100 100 100 100) 1 100 83 76 76 72 ND ND 2 79 76 72 68 68 64 5 83 74 74 72 65 65 23 75 72 72 64 66 66 100% activity of arginase is equivalent to 336 Iu/mg protein

5 Example 9A: In vivo half-life determination of arginase obtained from Example 8A Injecting poly(ethyl)-enzymatic acid into a patient. A 3 ml blood sample was taken from the string and in the EDTA every day. The tube was pre-cooled to 4 ° C on melted ice to prevent reaction from the body. The blood was then immediately spun at 14 rpm for 2 minutes to remove red blood cells. Aspirate 1.5 m of supernatant (blood) and 10 to a new microcentrifuge tube. The plasma was then incubated for 30 minutes at 37GC. After the incubation, arginine at a concentration of ΙΟΟμΜ was added as a substrate. The enzymatic reaction was carried out at 370C for 1 〇, 30 '60 minutes. At each time interval, the reaction was stopped by taking a 300 μl reaction sample and transferring it to a new microcentrifuge tube containing 300 μl of 1% trichloroacetic acid. Take the sample and rotate it at maximum speed (14000 rPm) for a minute. The supernatant was aspirated and filtered through a 0.45 μm filter. Finally, samples taken at different time intervals were analyzed using an amino acid analyzer (Hitachi, L8800). The results are shown in Fig. 21. Two batches of PEGylated arginase were prepared as described in Example 8A during the study. The first batch of pegylated arginase was prepared using a 1:140 molar ratio of sperm 41 1331531 lysinase: PEG. The second batch of PEGylated arginase was prepared using 丨:% ratio of arginase: PEG. The preparations and conditions for the preparation of the two batches of the product were the same (see Example - infusion at the time of 0 '50 mg of the first batch of polyethylene glycol servant &amp; and the spermatinase. 5 12 hours later, then again (four) Called PEG-argininase. The third arginase infusion was performed at 24 hours, using 50〇1§ first-batch alcohol arginase. From the 26th to the 72nd hour, Continuous infusion of the first batch of _ ^ a sterilized arginase (100 mg / day) instead of intermittent infusion (5 〇 mg / dose). From the first to the first 10 hours, 10 〇 mg / day continuous infusion of the second The PEGylated arginine enzyme was stopped at the first 44 hours, and continuous infusion of arginase was stopped. From this time point, the half-life was measured. The half-life measurement results are shown in Fig. 22. In Fig. 22, the time is equal to Figure 144, 144 hours. The results suggest that the half-life of arginase activity can be divided into two orders of 15 segments. The first half-life of PEGylase is about 6 hours. The relative activity is reduced from 100% to 50. % takes about 6 hours (see Figure 22). However, the second half-life is about 21 days. Relative activity 50% is reduced to 25°/. It takes about 21 days. This double half-life effect may be due to many factors, such as the use of higher amounts of mPEG-CC in PEGylation and the use of specific infusion protocols. Example 9B: Determination of the half-life of PEGylated arginase in vitro using a method in human plasma. Purified arginase (1 mg) was dissolved in ice and 125 mM borate buffer (pH 8.3). The activated PEg (mPEG-SPA, MW 5,000) (7.14 mg) was slowly added to the protein solution at a molar ratio of arginase:: 42 1331531 50. This mixture was prepared according to the method described in Example 8B. Stir on ice for 2.5 hours. Add t-ethylated arginase (305.6 μM) to human plasma (1 ml) at a concentration of 1 mg/ml, and the final concentration of PEGylated arginase is 〇24ing/mi 5 . The mixture was divided into 20 equal portions' placed in a microcentrifuge tube (65 μΐ in each microcentrifuge tube) and then incubated at 37 ° C. 1 - 2 μl mixture was taken from each microcentrifuge tube for testing of refined ammonia Acidase activity. The results are shown in Figure 2. The half-life was determined to be about 3 days. It takes about 3 days to reduce the activity from 1% to 50%. This is determined by the curve shown in Figure 20. 10 Example 10: B. subtilis-expressed human arginase and PEGylated Identification of isolated, purified and purified recombinant human arginase (4) arginase activity by jj_SDS-PAGE and photosynthetic sputum will be determined by Ikemoto et al. (ikem〇t〇 et al., 1990, Biochemistry 270, 697 -703) The purified E. coli expressed arginase obtained was compared with the purified human arginine spider expressed by the purified B. subtilis obtained by the method of the present invention (Fig. 19A and Fig. 19B). With Lumi-imagerTM

The Lumianalyst 32 program (Roche Molecular Biochemicals) performed a density analysis on the total protein bands shown in Figure 19A, indicating that the purity of the arginase produced by the method of the present invention was 99.9% or more (Fig. 19B). However, 80 - 2 〇 1% pure arginase can also be used as an active ingredient to prepare a pharmaceutical composition. In a preferred embodiment '80-100% pure recombinant arginase is used. In a more preferred embodiment, the recombinant arginase of the present invention has a purity of 90-1 〇〇〇/0 using SDS-PAGE followed by photoimage analysis. Fb) Determination of the rate of release of urea from L-arginine by arginase in a system by a coupling reaction ratio 43 1331531. The system contains urease, L-prolinamide dehydrogenase and NADPH (Ozer, R, 1985, Biochem. Med. 33, 367-371). To prepare the main mixture, 0.605 g of Tris, 0.0731 g of α-glutaric acid and 5 〇. 4355 g of arginine were dissolved in 40 ml of dH20. The pH was adjusted to 8.5 ' with 1 M HCl and then 〇. g 67 g urease was added to the mixture. The pH was further adjusted to 8.3 with HCl, followed by the addition of 0.0335 g of indoleamine dehydrogenase and 0.0125 g of NADPH. The final volume was adjusted to 5 〇 ml with dH20 to form a main mixture. This main mixture (1 ml) was drawn into a quartz cuvette. To measure arginase activity, '1-5 mg of arginase was added, followed by a 1-3 nm decrease in absorbance (Awe) at 3 °C for 1-3 minutes. The arginase of 1 I.U. is interpreted as the amount of enzyme that releases Ιμηηοΐ urea for 1 minute under the given conditions. The specific activity of the purified recombinant human arginase of the present invention was calculated to be 518 IU/mg protein, which was significantly higher than the reported activity of purified human erythrocyte arginase 15 (204 IU/mg protein; Ikemoto et al. 1989, Ann. Clin.

Biochem. 26 '547-553) and the activity value of isolated and purified recombinant human arginase expressed by E. coli (389 I_U./mg protein; Ikemoto et al., 1990, J. Biol. Chem. 270, 697-703). Using mPEG-SPA (MW 5,000), the PEGylated arginase retained as much as 114 °/ of the original activity at a 1:50 molar ratio and a reaction time of 2 hours 20 minutes and 40 minutes. Activity (see Example 8C). This means that the specific activity of the polyethylene glycolated human arginase is 592 I.U./mg. Using mPEG-CC (MW 5,000), PEGylated human arginase retains 64-68% of its original enzymatic activity (Table 3), similar to the pegylated bovine arginine 44 1331531 enzyme activity (Savoca, Κ. V. et al '1979, Biochimica et Biophysica Acta 578, 47-53). (V) Shooting natural arginase by derivatization LC/MS, 槿 槿 槿 枓 According to the amino acid sequence shown in Figure 2B, B_ subtilis-expressed and purified 5 recombinant human arginase contains 329 amino acid residues The theoretical molecular weight is 35, 647.7 Da. Simultaneous HPLC/UV and mass spectrometry analysis of the native arginase provides a molecular weight of 35,634 Da. The molecular weight of the observed natural arginase was found to be in complete agreement with the theoretical molecular weight of 35,647.7 Da from the expected amino acid sequence of the 6x histidine-tagged human arginase (Fig. 2B). The purity was found to be 98% by HPLC/UV detection based on LC/MS. The purity was found to be 1% by LC/MS based on HPLC/UV detection at 215 nm relative response. (d) by gel filtration chromatography on natural arginine decarboxylated PEGylation enzyme into samarium cobalt _ 枓 枓 in a HiLoad 16/60 superdex gel filtration column (Pharmacia) 15 'in pBS Gel transition chromatography was performed at a protein concentration of approximately 2-8 mg/ml in buffer. The molecular weight of the natural arginase was found to be approximately 78 kDa, and the molecular weight of the PEGylated arginase (prepared in Example 8C) was approximately 688. kDa. Since the molecular weight of the monomeric arginase is about 36 kDa, this result suggests that the natural arginase is present as a dimer in PBS buffer. 20 (e) Three-gas structure study The secondary structure of the purified arginase was analyzed in a JASCO J810 CD spectrometer using circular dichroism (CD). In the equivalent protein concentration in 10 mM potassium phosphate buffer (pH 7.4), when scanning from 195-240 nm, the CD spectrum of natural arginase and PEGylated arginase were found (in the implementation of 45 1331531 cases) The CD spectra prepared in 8C are very similar, indicating that the native form and the pegylated form of the arginase have nearly identical secondary structures. (f) pl point measurement Using a Bio-Rad 111 type small IEF battery, the isoelectric point (pi) of natural arginase 5 (prepared in Example 8C) was found to be 9.0, which is in comparison with the value published in the literature 9.1. Consistency (Christopher *Wayne, 1996, Comp. Biochem. Physiol. 114B, 107-132) 〇(g) Functional characterization and dynamic sputum determination using the method reported by Ikemoto et al. (1990, J. Biol. Chem. 270 10 ' 697- 703) Determination of arginase activity, natural arginase, 丨9 ± 0.7 mM 'Vmax is 518μπιο1 urea / min / mM ·, kcat is 2.0 soil 0.5 / sec, kcat / Km is 1 · 3 ± 0.4 / mM / sec. The !*^ value of the purified natural arginase was found to be very similar to the human liver arginase value (26 mM) published in the literature (Carvaja Buddhism et al., 1999). In addition, the maximum activity of the native arginine 15 enzyme requires approximately 1 mM of Μη2+ ions and 3〇~5〇. The temperature of 〇. The Km of the PEGylated arginase was 2.9 ± 〇.3, v a v max^ 360 μηιοί urea/min/mM. The value of PEGylated arginase is very similar to that of natural arginase, suggesting that the binding affinity of the enzyme to arginine is retained after PEGylation. In addition, achieving a maximum activity of PEGylated arginase requires about 1 mM of Mn2+ ions, a temperature of 4〇5〇〇c, and a pH of 10. The functional properties of arginase are similar before and after PEGylation, indicating that the covalent attachment of the mPEG-SPA molecule to the arginase generally improves its 46 1331531 nature. Example 11: Treatment regimen for exogenous administration of arginase A patient blood sample was taken daily during treatment to understand arginine levels, arginase activity, whole blood maps and coagulation maps. If you think you need it, test liver function and kidney function at least 5 every few days. Signs of life (BP, pulse, respiratory rate, blood oxygen readings) were measured every 15 minutes after the start of infusion of arginase for a total of 1 hour and then measured every hour until stable. After that, it is judged by the doctor. Twenty minutes before the infusion of arginase, 1 Omg of 10 dipheneramine was administered intravenously, and 1 mg of hydrocortisone was administered intravenously before each infusion of arginase or according to the judgment of the treating physician. The arginase was infused for 30 minutes on the first day. Thereafter, the arginase is infused weekly for at least 8 weeks. If anti-tumor activity is observed, an infusion can be continued. 15 Example 12: Example of a treatment regimen for exogenous administration of arginase In early August 2001, a 54-year-old Chinese woman was treated with a pegylated recombinant arginase, which had a metastatic rectum Cancer, extensively transferred to the lungs, all standard treatments are ineffective. The main symptoms are cough, loss of appetite and constipation. Its cancer marker CEA is 1100 U/ml. Treatment with PEGylated recombinant arginase was performed before treatment. Therapeutic Methodology 850 mg bed dry recombinant arginase I was administered. This drug was reconstituted in PBS and PEGylated. The PEGylase was found to have full activity. Results 47 1331531 The results are shown in Figures 28 and 29. Figure 28 shows that arginine was satisfactorily depleted to 1 - 5 μΜ over 5 days (see also Figure 21). Figure 29 shows that the CEA level has dropped from 1100 to 800 in 4 weeks. 1) Unlike chemotherapy, this treatment has no inhibitory effect on bone marrow and does not cause 5 hair loss. 2) This treatment can deplete arginine in a controlled manner. The arginine level is maintained within the therapeutic range for the desired 5 days (1_8 μΜ), and in vitro data provides the best tumor lethality. 10 3) No obvious side effects. The patient is tolerant of this treatment, but there is a dull pain, which may not be directly caused by treatment.

15 20

4) Biochemical and radiographic indications of the disease observed after treatment were all improved by a 30% reduction in CEA, and chest radiographs showed elimination of lesions in the upper region. Example 13: Treatment of laboratory rats with arginase for in vivo sperm depletion In this example, four groups of rats (two in each group, one male and one female), on the third day. Different enzyme 2: the arginase obtained from Example 8C on the first day before intraperitoneal injection of recombinant human arginase, on day I, then every two days, from large Blood samples were taken from the tail vein. . In Fig. 23, π' achieved arginine water in all test groups. It can be detected that the silk is dose-dependent and inhibits IU. After one day, arginine was depleted. Give UM morning to give Γυ / noon, ". to another 5GGLU_) 'There is 4 days of arginine completely depleted. Single; J 1500LU. There are 6 days of arginine depletion. Add the dose to 3 melon, refined ammonia The duration of complete acid depletion does not appear to be prolonged. Therefore, 'pegylated arginine 48 1331531, which is administered intraperitoneally, shows that it is the optimal dose for arginine depletion, and there is a 6-day arginine level. Not detectable. Example 14: Comparison of changes in blood component levels between normal rats and rats with zero arginine levels produced by arginase from day 1 to day 5 on day 0 Intracardiac arterial blood samples were taken from a group of 5 rats. The day of the day was used as an untreated control. Total protein, albumin 'globulin, SGOT/AST, SGPT/ALT, hemoglobin, fibrinogen Level, APTT·/sec, prothrombin/sec, white blood cell (WBC) and platelet count, determined by Pathlab Medical Laboratories, Inc., located at the second floor of Henan Building, 90-92 Jaffe Road, Wanchai, Hong Kong. Rat intraperitoneal injection of a dose of 1 500 IU arginase. Zero arginine levels were achieved in all rats. From day 1-5, one rat was sacrificed daily and intracardiac arterial blood samples were taken and determined by PathLab Medical Laboratory. The results confirmed by the laboratory showed that all proteins were within the normal range. 15 Example 丨5: Response to arginine depletion in nude mice bearing Hep3B tumors Human hepatoma cell line (Hep3B2. The right abdomen of 6 BALB/c nude mice was subcutaneously inoculated to grow the tumor. Three randomly selected mice were intraperitoneally administered with 500 IU of PEGylated arginase from Example 8C once a week. The other 3 mice were not given any arginase treatment as a control. The in situ growth of solid tumors in the implanted mice was determined every two days using a mathematical compass to determine the tumor size, calculated according to the following formula: Tumor size (mm) = average of two vertical diameters and one diagonal diameter. The number of deaths of mice in each group was also recorded every day. As shown in Fig. 24, in the first 20 days after the start of the experiment, the control group was 49 1331531 per day. Tumor size The growth rate was approximately 6 times higher in the PEGylated arginase treated group. 2 mice in the control group died within 24 days, while mice treated with PEGylated arginase survived at least 75. Example 16: Response to arginine depletion in nude mice bearing PLC/PRF/5 tumors 5 In this example, subcutaneous implantation of solid tumors of human hepatoma (PLC/PRF/5) The back of one BALB/c nude mouse was inserted to allow tumor growth. Five randomly selected mice were administered intraperitoneally once a week to 500 IU of PEGylated arginase from Example 8C, while others One mouse was intraperitoneally administered with 200 μM phosphate buffered saline (pBS) as a control. The in situ growth of solid tumors implanted in mice was measured every two days using a mathematical compass to determine tumor size and quality. Tumor size was determined as described in Example 15, and tumor mass was calculated according to the following formula: Tumor mass (mg) = length X width 2/2 (assuming specific gravity is 丨〇g/cm3) (where length is the longest vertical diameter, width is The shortest vertical diameter) 15 As shown in Figure 25A, 'in the first 39 days after the start of the experiment, the growth rate of the tumor in the control group was about 65 mm/day' in the PEGylated arginase-treated group. The 'tumor size growth rate is about 5.3 mm / day. As shown in Fig. 25B, the growth rate of tumor mass per day in the control group was about 1.8 times that of the treatment group. 20 Example 17. Response to arginine depletion in nude mice constituting HuH_7 tumors In this example, a solid tumor human hepatoma was implanted subcutaneously into the back of 10 BALB/c nude mice. To make the tumor grow. The randomly selected 5 〃, small milk was intraperitoneally-weekly administered with 5 〇〇··· PEGylated aptamase from the method described in the examples, and the other 5 mice were every 10 1331531 weeks. 200 μl of disc salt buffered saline (pbs) was administered intraperitoneally as a control. The in situ growth of solid tumors implanted in mice was determined by mathematical compasses every two days to determine tumor size and quality as described in Examples 15 and 16. As shown in Figure 26A, the growth rate of 5 tumors per day in the control group was approximately 6.0 mm/day during the first 18 days after the start of the experiment, and the growth of tumor size in the PEGylated arginase treatment group was increased. It is about 5 6] 11111 / day. As shown in the figure, the growth rate of the ribbed enamel # is about 1.4 times that of the treatment group. Example 18: Response to arginine depletion in nude mice of the MCF-7 axis of age 10 In &amp;Examples, human breast cancer cell line (MCF-7) was subcutaneously inoculated into 4 BALB/c The right abdomen of nude mice is used to grow the tumor. Three randomly selected mice were intraperitoneally administered with 500 I.U. PEGylated arginase from the method described in Example 8c one week, and the last one was administered without any arginase treatment as a control. The in situ growth of the solid tumor implanted in mouse 15 was measured every two days using a mathematical compass to determine the size of the tumor as described in Example 15. As shown in Figure 27, tumors inoculated with PEGylated arginase-treated mice disappeared within 20 days of the experiment. The singular forms 20, as used herein, and the terms used in the claims, "a," ", unless otherwise specified, "includes the plural. Thus, for example, a pharmaceutical product 'comprising a mixture of different articles, a method of treatment" includes equivalent steps and methods known to those skilled in the art, etc. Unless otherwise, all technical and scientific terms used herein have The same meanings are generally understood by those skilled in the art. Although any methods and materials similar or identical to those of 51 1331531 described herein can be used in the practice and testing of the present invention, preferred methods and materials are described herein. All publications in the specification are hereby incorporated by reference in their entirety for the entire disclosure of the disclosure of the disclosure of the disclosures of Modifications thereto are included in the scope of the present invention. The formulation of the pharmaceutical composition of the present invention may be used in the form of a solid, a wave, a milk wave, a dispersion, a capsule, a liposome or the like, wherein The resulting formulation in the practice of the invention contains one or more modified human arginase enzymes as a method 10 15 20

Sexual ingredients, mixed with an organic or inorganic body or excipient suitable for enteral or parenteral administration. The active ingredient may be an arginase, for example, f for use in monthly preparations, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for the production of solid, semi-solid or liquid form preparations. A non-toxic pharmaceutically acceptable carrier. In addition to the adjuvant, the counter% may be used in a stabilizer, a thickener and a pigment and a fragrance, and the amount of the arginase active ingredient contained in the drug is sufficient for the target process,

The disease produces the desired effect. Symptoms or diseases The pharmaceutical preparation containing the active ingredient of the present invention may be a suitable, and, in the form of a spoon, such as a tablet, a troches, a water-based or a Pei-wang problem; a float, a dispersed powder or granule, an emulsion , hard or soft capsules, biting sugar ' ' „10% or elixirs. Oral formulations can be prepared according to the production of pharmaceutical formulations known in the art. Tablets can be uncoated or can be packaged by known techniques Delaying disintegration and absorption in the gastrointestinal tract, thereby extending the effect over a longer period of time. They can also coat the osmotic agent to control the duration of 52 1331531. In some cases, the oral formulation can be a hard gelatin capsule. The active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, kaolin, etc. They may also be in the form of a soft gelatin capsule in which the active ingredient is mixed with a water or oil medium such as peanut oil, liquid paraffin or olive oil. The formulation may also be a sterile injectable solution or suspension. The suspension may be formulated according to known methods using a suitable dispersing or moisturizing or suspending agent. The bacterial injection preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,4-butanediol. A sterile, non-operable oil is conventionally used as a solvent or 10 Suspension medium. Any soft, non-volatile oil can be used for this purpose, including synthetic mono- or diglycerides, fatty acids (including oleic acid), natural vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil, or synthetic A fatty acid carrier such as ethyl oleate, etc. If necessary, a buffer, a glucose solution preservative, an antioxidant or the like may be incorporated or they may be used as a solute 15 to dissolve a soluble enzyme. The pharmaceutical formulation may also be used as an adjuvant treatment. The agent is used together with other chemotherapeutic agents. In the claims, an arginase having an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID No. 9 means that the sequence is as shown in SEQ ID No. 9. The sequence is at least 30% identical, or the enzyme shown in SEQ ID No. 9 and the enzyme substantially similar thereto are shown in the enzyme using the arginase activity assay described herein. There is no significant difference in sex. Six histidines are provided for ease of purification, and additional methionine groups are provided at the amino terminus for translation initiation. Those skilled in the art understand that other forms of purification can be used 53 1331531 Thus, the "substantially similar" arginase does not require any homology to the sequence of SEQ ID No. 3. In some bacterial strains, there may be at least 40% homology to SEq SEQ ID No. 9. Some mammalian arginase enzymes may have at least 70° homology to SEQ ID No. 9. 5 Sequence Listing SEQ ID NO: 1 (Figure 2A) SEQ ID NO: 2 &amp; 3 (Figure 2B: Nucleotide sequence ( SEQ ID NO: 2); and amino acid sequence 歹|J (SEQ ID NO: 3)) SEQ ID NO: 4: 6xHis tag MHHHHHH 10 SEQ ID NO: 5: 5, -CCAAACCATATGAGCGCCAAGTCCAGAACCATA-3, (Arg 1) SEQ ID NO: 6: 5, -CCAAACTCTAGAATCACATTTTTTGAATGACATGGACAC-3 , (Arg 2) 15 SEQ ID NO: 7 : 5, -CTCTGGCCATGCCAGGGTCCACCC-3, (Arg 6) SEQ ID NO: 8 &amp; 9 : (2C: nucleotide sequence (SEQ ID NO: 8); amino acid Sequence (SEQ ID NO: 9)). I: Schematic description of the figure 3 Fig. 1 shows a plasmid map of ρΑΒΙΟΙ. This plasmid carries the gene (arg) encoding the arginine 20 acidase and is only replicated in E. coli and not in B. subtilis. 2A and 2C show the nucleotide sequence of human arginase I and its deduced amino acid sequence. Figure 2A shows the nucleotide sequence of the EcoRI/MunI to Xbal site of the plasmid pΑΒΙΟΙ (SEQ ID NO: 1). Nuclei 54 1331531 Glycosyl (nt) 1 — 6, EcoRI/MunI site; nt 481 — 486, promoter-35 region; nt 504-509, promoter 1-10 region; nt 544-549, promoter -35 region of subunit 2; nt 566-571, region-10 of promoter 2; nt 600-605, ribosome binding site; nt 614-616, initiation codon; 5 nt 632-637, Ndel locus ;nt 1601 — 1603, stop codon; nt 1997 — 2002, Xbal locus. Figure 2B shows the nucleotide sequence encoding the modified human arginase (SEQ ID NO: 2) and its corresponding encoded amino acid sequence (seq id NO • 3). Nucleotides 616-1603 in Figure 2A are the coding regions for the modified arginase amino acid 10 sequence. The 6-histidine (SEQ ID NO: 4) label at the end of the sputum is underlined. The translation stop codon is indicated by *. Figure 2C shows the nucleotide sequence encoding the normal human arginine (seq IDNO: 8) and its corresponding encoded amino acid sequence (seqidn〇: %. Figure 3 is the construction of B expressing arginase) Schematic diagram of s protoplasts. Figures 4A and 4B show the fermentation time course of recombinant Bacill-strain LLC (8) in a 2 L fermentor. Figure 4A shows the results obtained from sub-wholesale. The result of fed-batch fermentation. 20

Figure 5A and the second paste show the fermentation history, showing changes in parameters such as degree 'na speed, pH and dissolved oxygen values. Figure 5a shows the history of the wholesale. Figure 5B shows a history of fed batch fermentation. Fig. 6A and Fig. 6B show the results of a biochemical purification after the first 5 ( (4) human arginine _ for 3 hours after the hot pier, and the operation parameters and egg (four) washing (4) The first steel shows; 55 1331531 SDS_pAGE (12%) analysis of 5 μ 1 11 - 31 fractions collected in the column. The protein gel was stained with Coomassie brilliant blue and decolorized to show the protein band "lane M: low molecular weight range label (1 μ§/band; Bi〇_Rad) 'MW (Dalton): 97,400; 66,200 45,000; 31,000; 21,500 5; 14,400. 10 15 20

Figures 7A and 7B show the results of purification of human arginase by a second 5 ml H^Trap binding column 3 hours after heat shock. Figure 7A shows FPLC operating parameters and protein elution profiles. Figure 7B shows an SDS_page 〇 2% analysis of 1 μ19-39 fractions collected from the column. The protein gel was color-coded with Coomassie and decolorized to show the protein bands. Lane ^[: low molecular range § hex (1 gg / band; Bi 〇 _Rad), MW (Dalton) ·, 66, 200; 45, 0 〇〇; 31, 〇〇〇; 21,500; 14,400. Figures 8A and 8B show the results of purification of human arginase by the first 5 ml HiTrap gel column 6 hours after heat shock. Figure 8A shows

FPLC operating parameters and protein elution profiles. Figure 8B shows an SDS-PAGE (12%) analysis of 2.5 μl 1 〇 -32 fractions collected from the column. The protein gel was stained with Coomassie Brilliant Blue and destained to show the protein band.泳 Μ low knife i range standard redundancy (1 call / strip; Bi〇_Rad), mw (Dalton) .97,400, 66,20〇; 45, 〇〇〇; 31,000; 21,500; 14,400 . Fig. 9A and Fig. 9Bg] show the results of purification of human arginase by a second twisting &amp; kneading column 6 hours after heat shock. Figure 9A shows the d FPLC operating parameters and protein elution profiles. The figure shows the SDS-PAGE (12%) analysis of the fractions from the column | W 8 to E6. Protein condensate was stained and discolored with the Coomassie field to show the egg self-filament. Lane M: Bo 56 1331531 Molecular weight range label (1 gg/band; Bio-Rad) 'MW (Dalton): 97,400; 66,200; 45,000; 31,000; 21,500; 14,400. Figure 10 shows the time course of bacterial cell growth when heat shock is performed at a higher cell density. When the culture density (OD6 〇 Gnm) was about 25, 5 heat shock was performed at 8 hours. Figure 11 史 ^ History of fed-batch fermentation in the heat of the southern male. This graph shows parameter changes such as temperature, picking speed, pH and dissolved oxygen values. Fig. 12A and Fig. 12B show the results of purification of human arginase by the first 5 ml HiTrap integration column after 6 hours of heat shock (at a higher cell density of 25). Figure 12A shows FPLC operating parameters and protein elution profiles. Figure 12B shows the results of SDS-PAGE (12%) analysis of 5 μM of each fraction collected from the column. The protein gel was stained with Coomassie brilliant and decolorized to show the protein bands. Lanes Μ: low molecular weight range 15 markers (1 gg / band; Bio-Rad), MW (Dalton): 97, 40 〇; 66 200, 45,000, 31,000, 21,500; 14,400. Lane "crude extract". 5 μΐ of the crude cell extract before being added to the column. Fig. 13 and Fig. 13 show the results of purification of human arginase 20 by a second 5 ml HiTrap 6 6 6 hours after heat shock (higher cell density at 〇D25). Figure 13A shows the FPLC operating parameters and protein, and the first release. Figure 13B shows the results of SDS-PAGE (12%) analysis of 7 μ% of each fraction collected from the column. The protein gel was stained with Coomassie Blue and destained&apos; to show the protein bands. Lane Μ: marker of low molecular weight range (1 μ§/band; Bio-Rad), MW (Dalton): 97,4〇〇. 66 mo 57 1331531 ‘· 45,000; 31,000; 21,500; 14,400. Figures 14A and 14B show the results of purification of human arginase by the first lml HiTrapSP FF binding column 6 hours after heat shock (higher cell density at 〇D25). Figure 14A shows FPLC operating parameters and protein elution profiles. Figure 14B shows the results of SDS-PAGE (12%) analysis of 5 μM of each fraction Α11 - Β7 collected from the column. The protein gel was stained with Coomassie brilliant blue and destained to show the protein bands. Lane Μ: marker of low molecular weight range (1 pg/band; Bio-Rad), MW (to Dalton): 97, 4〇〇; 66, 200; 45,000; 31,000; 21,500; 14,400. 10 Figures 15 and 15B show the results of purification of human arginase by a second lml HiTrap SP FF binding column 6 hours after heat shock (higher cell density at OD25). Figure 15A shows the FPLC operating parameters and the protein elution profile. Figure 15B shows the results of SDS-PAGE (12%) analysis of 5 μM of each fraction Α6 - Β12 collected from the column. The protein gel was stained with Coomassie Brilliant Blue and destained to show the protein bands. Lane Μ: low molecular I range marker 〇Hg/band; Bio-Rad), MW (Dalton): 97,400; 66,200; 45,000; 31, 〇〇〇; 21,500; 14,400 = Figure 16A and Figure 16B The ratio of arginine 2 tannase to PEG used for 8 〇 3_1 &gt; eight (}] 5 (15%) analysis of human arginase modified with mPEG-SPA (MW 5, 〇〇〇) 1 : 50. Figure 16A shows the results when the reaction was carried out on ice. Lane 1: low molecular weight range protein labeling; lane 2: arginase without PEG (5.35 pg) (control); lane 3 : 1 hour after the reaction 'lane 4: 0.5 hours after the reaction; lane 5: 2 hours after the reaction; lane 6 · 3 hours after the reaction; lane 7: 4 hours after the reaction; lane 8: 58 1331531 after the reaction 5 hours; : 23 hours after the reaction. Figure 16B shows the results of the reaction at room temperature. Lane 1: low molecular weight range protein labeling; lane 2: arginase without PEG (5.35 pg) (control); lane 3 : 1 hour after the reaction; Lane 4: 0.5 hours after the reaction; Lane 5: 2 hours after the reaction; Lane 6 5: 3 hours after the reaction; Lane 7: 4 hours after the reaction; Lane 8 : 5 hours after the reaction; Lane 9: 23 hours after the reaction. Figures 17A and 17B are SDS-PAGE (15%) analysis of human arginase modified with mPEG-SPA (MW 5,000), used The ratio of arginase to PEG is 1 ··20. Figure 17A shows the results when anti-10 is performed on ice. Lane 1: low molecular weight range protein label; lane 2: arginine without PEG added Enzyme (5.35 pg) (control group); Lane 3: 1 hour after reaction; Lane 4: 0.5 hour after reaction; Lane 5: 2 hours after reaction; Lane 6: 3 hours after reaction; Lane 7: 4 hours after reaction; Lane 8: 5 hours after the reaction; Lane 9: 23 hours after the reaction. Figure 17B shows the results of the reverse reaction at room temperature. Lane 1: low molecular weight range protein labeling; lane 2: arginine without PEG added Enzyme (5.35 gg) (control group); Lane 3: 1 hour after reaction; Lane 4 · 0.5 hours after reaction; Lane 5: 2 hours after reaction; Lane 6: 3 hours after reaction; Lane 7: 4 hours after reaction Lane 8: 5 hours after the reaction; Lane 9: 23 hours after the reaction. 20 Figure 18A shows human arginine modified with mPEG-CC (MW 5,000) SDS-PAGE (15%) analysis was performed. The reaction was carried out on ice. Lane 1: low molecular weight range protein labeling; lane 2: arginase without pEG (5.35 pg) (control); lane 3: 1:50 molar ratio of arginase: 2 hours after PEG reaction; Lane 4: blank; Lane 5: 1:50 59 1331531 molar ratio of arginase: 23 hours after PEG reaction; Lane 6: used 1: 20 molar ratio of arginase: 2 hours after PEG reaction; Lane 7: 1 • 20 molar ratio of arginase: 5 hours after PEG reaction; Lane 8: using 1·20 molar ratio of refined ammonia Acidase: 23 hours after PEG reaction. 5 Figure 18B shows an SDS-PAGE (12 ❶/.) analysis of natural arginase and PEGylated arginase, which are highly active and stable. Lane 1: low molecular weight range protein labeling (Bi〇_rad); lane 2 • natural arginase (1 Mg); lane 3: PEGylated arginase (1 pg); lane 4: after ultradialysis Pegylated arginase 〇.5 μβ). 10 Figure 19 and Figure 19 show the determination of the purity of the isolated recombinant human arginase. Figure 19 shows Lane 1: 5 pg of purified E. coli expressed recombinant human arginase obtained from the method described by Ikemoto et al. (Ikemoto et al. 1990, J. Biol. Chem. 270, 697-703). Lane 2: 5 pg of purified recombinant human arginase expressed by B. subtilis from the method of the invention. Figure 19B shows the density analysis of the protein bands shown in Figure 19A using the Lumianalyst 32 program of Lumi-imagerTM (Roche Molecular Biochemicals). Top: Results of Lane 1 of Figure 19A. Below: Results of Lane 2 in Figure 19A. Figure 20 is a graphical representation of the in vitro stability of PEGylated arginase in human plasma. Figures 21 and 22 show the half-life determination of the poly(ethylene glycol) arginase from the method described in Example 8A in vivo. Figure 21 shows the in vivo activity of PEGylated arginase produced by the method of the present invention as measured using the activity assay described in Example 9A. 60 1331531 Figure 22 is a graphical representation of the first half-life and the second half-life of a PEGylated arginase. Figure 23 is a comparison of arginine depletion in four groups of laboratory rats administered intraperitoneally with different doses of PEGylated recombinant human arginase 5 (500 IU, 1000 IU, 1500 IU and 3000). IU). Fig. 24 shows a comparison of survival rate, mean tumor size, and tumor growth rate of two nude mice which were tumor-produced by implantation of Hep3B cells. One group was treated with an intraperitoneal administration of 500 I.U. of arginase and the other control group was not treated with arginase. 10 Figures 25A and 25B show the mean tumor size and the mean tumor weight comparison of two groups of nude mice which were tumorigenic by implantation of PLC/PRF/5 cells. One group was treated with an intraperitoneal administration of 500 I.U. arginase and the other control group was not treated with arginase. Figures 26A and 26B show the mean tumor size and the mean tumor weight comparison of two groups of nude mice which were tumorigenic by implantation of HuH-7 cells. One group was treated with an intraperitoneal administration of a 500 I.U. dose of arginase, and the other control group was not treated with arginase. Figure 27 shows the average tumor size comparison of two groups of nude mice which were tumorigenic by implantation of MCF-7 cells. One group was treated with an intraperitoneal administration of 500 I.U. 20 doses of arginase and the other control group was not treated with arginase. Figures 28 and 29 show the arginine and CEA levels in the patient during treatment as described in Example 12, respectively. [The main components of the diagram represent the symbol table] None 61

Claims (1)

1331531 Policy _9.2L24.884 - No. 4f: Application for patent 蓺圚绦正太99年月年月吏) Original - 1. A covalent attachment of the coupling agent amber sulphonic acid (SPA) Recombinant human arginase I of polyethylene glycol having an in vitro plasma half-life of at least three days and a specific activity of 250 - 592 IU/mg, wherein the recombinant human 5 arginase I is encoded as a nucleotide Sequence (SEQ ID NO: 8). A pharmaceutical composition comprising a recombinant human arginase I as in claim 1 of the patent application. 3. The pharmaceutical composition of claim 2, wherein the composition is further formulated in a pharmaceutically acceptable carrier. 10. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is in a form suitable for oral administration, sterile injectable solution or sterile injectable suspension. 5. A pharmaceutical composition according to claim 4, wherein the recombinant human arginase I has a half-life of at least 3 days in the plasma of the patient. 15 6. A recombinant human arginase I is produced as in claim 1 Prepare a drug for use. 7. The use of claim 6 wherein the medicament is for the treatment of a human malignancy. 8. The use of claim 7 wherein the human malignancy 20 is a liver tumor or breast cancer.