CN102552933A - A chemically modified methioninase and use of the same in antimethionine and anti-homocysteine chemotherapy - Google Patents

Abstract

The invention provides a chemically modified methioninase and use of the same in antimethionine and anti-homocysteine chemotherapy. The invention provides recombination host cell including an expression model of encoded chemically modified methioninase. The expression model includes a nucleotide sequence of the chemically modified methioninase connected to a subsequence through an encoding operation. The starting of the subsequence can lead the expression of the chemically modified methioninase in the hose cell. And the expression of the chemically modified methioninase is 5 to 75 percents of total protein generated by the host cell. The invention further provides a method for producing the total protein and a method for identifying the recombination host cell having the chemically modified methioninase in a high content.

Description

The application of methioninase in the chemotherapy of anti-methionine and anti-homocysteine

The application be that June 7, application number in 1996 are 200610099783.9 the applying date, denomination of invention divides an application for the application of " application of methioninase in the chemotherapy of anti-methionine and anti-homocysteine ".

Technical field

The present invention relates to the methioninase compositions, the purification process of methioninase is attached to the method for polymer such as PEG (Polyethylene Glycol) to methioninase, the method for use methioninase in the chemotherapy of anti-ammonia egg acid and anti-homocysteine.In particular, method of the present invention comprises methioninase in treatment of cancer, the use in cardiovascular disease therapies and tumor imaging and the diagnosis.The invention still further relates to recombinant DNA technology.The present invention provides the expression model of coding methioninase especially.

Background technology

Be that main cancer therapy is meant that use can selectivity suppresses or kills cancerous cell with the medicine, but the function of normal structure do not caused the medicine of the damage that exceeds acceptable scope.The difficult point of routine treatment is the toxicity of medicine to normal structure.

Have now found that many tumors all have absolute demand to methionine in various cell types and the tumor tissues commented on.Wherein said cell and tumor tissues comprise tumor and leukemia and the lymphoma in colon, mammary gland, prostate, ovary, kidney, larynx melanoma, sarcoma, lung, brain, the harmonization of the stomach bladder.Clearly when the methionine in the growth medium is replaced by homocysteine, the no energy for growth of tumor has been defined as the dependency to methionine at present.For example, referring to Chello etc., Cancer Res., 33: 1898-1904,1973; And Hoffman, Anticancer Res., 5: 1-30,1985.

Shown that at present removing methionine (methionine depletion) can optionally make the tumor cell that relies on methionine get into the S/G in this cell cycle synchronously ₂Late period, Hoffman etc., Proc.Natl.Ad. Acad.Sci.USA.77: 7306-7310,1980.Unite to use and remove methionine; Then and be exposed to antimitotic agent; Be the chemotherapy of anti-methionine, tumor cell optionally removal from the coculture of normal and tumor cell, the culture that the result causes the normal cell cell is hypertrophy forcefully.Stern etc., J.Natl.Cancer Inst., 76: 629-639,1986.

But; In order to carry out methionine dependency chemotherapy in vivo; A kind of method of from serum, effectively removing the circulation methionine must be arranged; Also nobody described a kind of like this method of removing methionine at present, and it is effective that the content that promptly this method can reduce the methionine of body-internal-circulation is enough in antineoplaston it.

Be purified into a kind of enzyme that can degrade methionine from various bacterial origins is methioninase at present, and it is reported that this kind of enzyme can slow down the outgrowth speed of tumor cell in vitro.Kreis etc., Cancer Res., 33: 1862-1865 and 1866-1869,1973; Tanaka etc., FEBS Letters.66: 307-3111976; Ito etc., J.Biochem., 79: 1263-1272,1976 with Nakayama etc., Agric.Biol.Chem., 48: 2367-2369,1984.

Kreis etc., Cancer Rs., 33: 1866-1869; Described in 1973 and used isolated very impure methioninase preparation from clostridium sporogenes (Clostridium sporgenes), suppressed to move into the growth of the carcinosarcoma cell in the mouse model with the amount in 1150 units/kg/skies.Although this enzyme has obviously reduced the growth of primary tumo(u)r cell, reported never that leading the T/C of diameter of tumor (handling the ratio with contrast) was reduced to below 50%, and reported never also that metastatic tumor was had any effect.The author also points out not have other nonspecific interference can not predict the tumour-specific of methioninase, and the author does not comment on endotoxin in the pure preparation not or other components probability to viewed effect role yet.Unique toxicological study of being reported is that the weight of animals does not alleviate and toxic general macroscopy is negative after the treatment phase.In addition, the author has reported that this kind of enzyme has 4 hours serum half-life.This kind of enzyme preparation has only half purity and contains the endotoxin of significant quantity, and this makes complicated to this result's explanation.

And Kreis can not be reduced to below 8% of matched group to the content of serum methionine, and this maybe be owing to the high K of bacillus (Clostridium) enzyme _m(approximately 90mM).It is reported that the methioninase preparation is unstable and in to its purification, can only obtain 2% recovery (productive rate).

Kreis etc., CancerRes., 33: 1866-1869, also reported that a kind of antineoplastic method promptly uses the food that does not contain methionine as the method for removing methionine in 1973.But the author reports that this way of dining can not be as the growth of using impure methioninase preparation to slow down cell effectively, and can cause the weight of animals this side effect that occurs do not hoped that continues to descend.The author does not report that the associating use does not contain the food and the methioninase treatment of methionine, and does not study the synchronization of cell.

Application formerly of the present invention has proposed effective tumor chemotherapy, thereby these chemotherapy are meant the content that uses methioninase effectively to reduce methionine a kind of undamaged antineoplastic beneficial effect is provided.The present invention has proposed treatment and diagnostic method and compositions through providing a kind of high-purity RMETASE that is used to produce commercial feasible number to improve these applications.

Summary of the invention

Have now found that; Methioninase; No matter be that the form of PEGization (PEGylated) or produce do not contain endotoxic recombinant forms with purification highly purified, can both remove the intravital methionine content of mammal harmlessly, can both be used for selectivity effectively and suppress growth of tumor; And can optionally prevent tumor cell, make itself and antimitotic chemotherapy synchronization.

Find also that now methionine removal method is the most effective when quite a few kinds of diverse ways of collaborative use reduce the content of methionine basically effectively.These methods comprise that use does not contain the methionine starvation method of the diet of methionine; Use can be competed the methionine competitive inhibition method that methionine utilizes the inhibitor of enzyme (methionine-utilizing enzyme) with methionine, is beneficial to the chemotherapeutant that specific tumors selecting cell cycle of being brought out by methionine removal method prevents and the combination of these methods.

So; The invention describes a kind of method that suppresses growth of tumour cell; It comprises and contacts one period that is enough to bring out the cell cycle arrest of cell in the cell mass to tumor cell group with the methioninase defined herein of treatment effective dose, and forms immobilized tumor cell." contact " is to such an extent as to a speech is meant puts therapeutic combination of the present invention and target tumor very near observing the effect that suppresses tumor; Perhaps be placed on therapeutic combination of the present invention in a kind of culture medium such as Nutrient medium or the blood, to such an extent as to can make the methionine content in the culture medium be reduced to the level that can suppress the growth of tumour cell in the culture medium." immobilized " tumor cell is meant the downtrod tumor cell of growth.This method can be used in external or body.Need not be directly with contacting of tumor cell, it can be in external or body contacts with the Nutrient medium that contains tumor cell realizes.This contact also can contact blood circulation and accomplish with methioninase.When tumor is that this blood circulation can contain tumor cell or not contain tumor cell can not the circulation solid tumor time." suppress the growth of tumor cell " and be meant that taking The compounds of this invention can reduce growth of tumor; For example weigh with volume; With do not compare with the tumor of this compounds for treating; Tumor size can reduce by 10% to 100%, and more preferably at least 34% to 79%, most preferably be and be reduced to 55% to 68%.

Methioninase is used as and slows down or stop fissional a kind of antitumor agent through removing methionine.Use the competitive inhibitor of methionine can strengthen this effect.In addition, use methioninase can increase the treatment effectiveness of the specific cell toxin agent of antimitotic agent or other cell cycles with the specific cell toxin agent of antimitotic agent and other cell cycles through bringing out the cell cycle synchronization.

The chemotherapy that methioninase also can be used for removing homocysteine reduces the danger and treatment cardiovascular disease of cardiovascular disease.When methioninase also is used for detection of superelevation resolution ground and diagnosing tumour, sufficient again the confession [ ¹¹C] remove before the methionine in blood and the tumor [ ¹²C] methionine.

In a technical scheme, at first make tumor cell accept the content that the hungry step of methionine reduces first methionine.Can follow the egg starvation method to reduce with height half dirty propylhomoserin without restriction, thereby increase the specificity of treatment Normocellular toxicity.

The characteristic of other related fields of the present invention is to use methionine removal method of the present invention that tumor cell is prevented the S/G2 late period in cell cycle, and the agent of specific cell toxin such as the antimitotic agent that then begin the processing of cell cycle synchronously and take a kind of cell cycle to kill optionally and effectively tumor cell.Those of ordinary skills know the concrete stages of cell of cell cycle and have toxic cell cycle specific cytotoxic agent.This method comprises the following steps:

(a) the immobilized tumor cell of removing generating step by above-mentioned methionine contact with the methionine of the consumption that can bring out cell cycle, with the cell cycle that starts the cell (cycling-cells) that the static tumour cell cycle of formation grows and

(b) cell of cycle growth is contacted with the specific cell toxin agent of a certain amount of cell cycle.The cytotoxic agent consumption of this cycle growth will be enough to suppress the mitosis of mitotic cell, suppresses the growth of tumor cell thus.

In a technical scheme, this method comprises the following steps:

(a) the static tumor cell of removing generating step by above-mentioned methionine is contacted with the methionine that can bring out the cell cycle consumption start the mitosis that a cell is arranged that forms static tumor cell and

(b) mitotic cell is contacted with a certain amount of antimitotic agent, this antimitotic agent consumption will be enough to suppress the mitosis of mitotic cell, suppresses the growth of tumor cell thus.

According to an aspect of the present invention, a kind of method of tumor of diagnosing patients is provided, has comprised: a) to remove the patient intravital through take methioninase to the patient ¹²The C methionine; B) through taking to the patient ¹¹The C methionine comes to ample supply methionine in patient's body; And c) exists in the mensuration patient interior tumor cell ¹¹The level that the C methionine raises.

Another characteristic of the present invention is to use methioninase to reduce the dangerous of cardiovascular disease and treat cardiovascular disease to reduce the intravital cysteine levels of patient.

The invention provides through the administration methioninase to remove the method that methionine carries out diagnosing tumor and radiography.

Another characteristics are to be used for the therapeutic combination that methionine is removed therapy; It contains the isolated basically methioninase of treating effective dose; The specific activity that this methioninase has is that methioninase activity and every milligram of albumen of every milligram of about at least 10 to 40 units of (mg) albumen is less than about 1 to 100ng endotoxin; In preferred scheme, this therapeutic combination comprises that every milligram of albumen is less than the endotoxin of 10ng, with a kind of pharmaceutically suitable carriers.

Therapeutic combination of the present invention also comprises and do not contain endotoxic methioninase, in a technical scheme, methioninase be to use a kind of new effective method from the pseudomonasputida of growing down at the fermentation condition of improvement ( Pseudomonas putida) in make.The present invention also provides a kind of method of pseudomonas putida bacterial strain of High-efficient Production methioninase of separation improving in this respect.In another scheme, methioninase is to be produced by the recombinant host of the expression model that contains the methioninase of encoding.

Methioninase compositions of the present invention also can provide with a kind of form of chemical modification through methioninase being coupled to polymer such as Polyethylene Glycol (PEG).The methioninase that the PEG that the present invention uses modifies has the activity of efficient removal methionine, the half-life of prolongation and reduced immunogenicity.

Methioninase compositions of the present invention also can utilize the form of the recombiant protein that a kind of expression vector such as efficient expression vector defined herein produce to provide.Employing efficiently expresses the methioninase that system produces and can use method described herein to come purification to obtain not contain endotoxin and very pure methioninase compositions at an easy rate, and said composition has the specific activity of about 10 to 40 unit/milligram methioninases.

The present invention also provides to contain and can express the high carrier that gets the nucleic acid of unusual high-load methioninase.Now used this carrier such as those to use the carrier of T7 rna polymerase promoter; Under suitable incubation conditions, produce about 1 to 4 gram methioninase/liter methioninase; Its specific activity in rough tissue homogenate is about 2.6 to 5, can be expressed as 10% to 20% of about total cell protein.

The present invention also provides gene therapy method, and use promptly as herein described can be used for substituting the method for the clone's contain the methioninase compositions methioninase gene.The intravital cell of patient can be changed expresses methioninase provides therapeutic dose for the patients serum methioninase.

The invention still further relates to isolated nucleic acid molecule, the nucleotide sequence that contains among Fig. 8 to be provided and one or more can be at the C-of methioninase or the nucleotide sequence of the two or more histidine residues of N-end coding.

The invention still further relates to and use a kind of gene therapy of expressing the improvement of model.

Other characteristics of the present invention, advantage and relevant programme will be main the elaboration with the description that this paper was comprised.

Summary of the invention

A. Definition

" amino acid residue " is meant the formed aminoacid of chemical digestion on the polypeptide peptide chain (hydrolysis) polypeptide.Amino acid residue as herein described refers in particular to " L " optical isomer form.But as long as polypeptide has kept required functional characteristic, the residue on " D " optical isomer can be replaced by any L-amino acid residue.NH ₂-be meant to be present in the N-terminal free amine group of polypeptide.COOH is meant the free carbonyl that is present in polypeptide carbonyl end.This paper kept standard the polypeptide nomenclature ( J.Biol.Chem., 243: and 3552-59,1969 is described, and by 37C.F.R.1.822 (b) (2)) adopt, be cited as reference paper here.

It should be noted that it all is orientation from left to right to the general direction of c-terminus that this paper uses the aminoterminal of all represented amino acid residue sequences of general formula.In addition, widely be defined as phrase " amino acid residue " and comprise aminoacid listed in the aminoacid table and improvement and aminoacid that be of little use, as list in this paper and be cited as those aminoacid among the 37CER 1.822 (b) (4) of reference paper.And, should be noted that at amino acid residue sequence initiating terminal or terminal short-term and represent to the peptide bond of another one or a plurality of amino acid residue sequence or to aminoterminal group such as NH ₂Or the covalent bond of acetyl group or c-terminus group such as COOH.

" recombinant DNA (rDNA) molecule " refers to through operability and connects two formed dna moleculars of dna fragmentation.So recombinant DNA molecules is the dna molecular that contains the hybridization of at least two nucleotide sequences that common discovery is not together in nature.Do not have identical biogenetic i.e. evolution to go up different rDNA and be called " allogenic " rDNA.

" carrier " refers in cell rDNA molecule that can self-replicating and the section that adheres in order to make duplicates the rDNA molecule of can be operably connected DNA section such as gene or polynucleotide.This paper expresses the gene of the one or more polypeptide of coding leading carrier is called " expression vector ".The carrier of particular importance can easily be expressed methioninase albumen of the present invention.

B. use the method for methioninase as antitumor agent

I. The removal of methionine

This paper uses methioninase to come from tumor cell, tumor tissues as antitumor agent in every way or suffers from and remove methionine the mammiferous blood circulation of cancer basically or need to remove under the situation of methionine the removal methionine at thinking of as this paper below, being described in detail.

Although existent method can be reduced to intravital methionine content and be not less than about 35 μ m, also do not have at present a kind ofly can be reduced to the method that almost is lower than about 10 μ m to methionine content safe, easy and rapidly.Basically be meant that the conventional determining method that uses methionine can record methionine content at least and be lower than 10 μ m, preferably be less than 1 μ m, more preferably be less than 0.1 μ m, preferably can not measure the content of methionine.Use certain methods to comprise in body fluid such as blood, blood plasma and the serum determining methionine at aqueous solution.An illustrational method is to use the anti-phase FPLC method of methionine reference material.

In vivo; In the mammiferous circulation; Need remove under the vitro conditions of methionine at tissue culture or other biological culture medium, and at manipulation in vitro biological fluid, cell or tissue and then put back in the intravital stripped method of mammalian subject and can both remove.

The removal of from circulation, culture medium, biological fluid or cell, carrying out methionine is to want the amount of the methionine of therapeutant in order to reduce entering; Therefore it is included in following the material that will remove of condition of removing methionine and contacts with the methioninase of the present invention of removal methionine amount, degrades by the methionine in the contact material.

Because tumor cell depends on the methionine in their Nutrient medium, thus can remove the nutrient source of these cells, and needn't remove these cells itself.Therefore, use the present invention in vivo, methioninase is contacted with the nutrient media of tumor cell group.In this scheme, medium can be the body fluid that blood, lymph fluid, cerebrospinal fluid etc. need to remove methionine.

The amount of removing methionine can change in wide range according to the difference of using, and depends on that generally the amount of the methionine that exists in the material, required removal speed and this material are for the toleration that is exposed to methioninase.Use various chemistry known in the art and biochemical method can monitor the content of the methionine in the material at an easy rate and from this material, remove the speed of methionine.This paper has also described the example of removing the methionine amount, wherein is processed in the material at every milliliter, and the scope of methioninase is 0.001 to 100 unit (U), preferably approximately is 0.01 to 10U, and more preferably about 0.1 changes in the scope of 5U methioninase.

The condition of removing methionine is buffer and the temperature conditions that the biological activity with methioninase adapts, and comprises demulcent temperature, salt and the pH condition that adapts with this enzyme.Although preferred physiological condition, the example of condition comprise 4-40 degree centigrade (℃) about, the ionic strength with respect to 0.05 to 0.2M NaCl and about 5 to 9 pH.

In a preferred scheme; The present invention has imagined the method for methioninase as antitumor agent of using; Contact one period that is enough to bring out the cell cycle arrest of the cell in the colony to tumor cell group with the methioninase of treatment effective dose so it comprises, and form immobilized tumor cell.

As described herein; Because a variety of causes; Include but not limited to; Slow down optionally that growth of tumor, the period through keeping the cell of lag phase one elongated segment are killed tumor cell with the method that produces cell death and before the chemotherapy of following, be that the cell cycle synchronization prepares tumor cell group etc., all need make tumor cell be in the cell cycle arrest phase.

Bring out the required time of cell cycle arrest and depend on quite a few kinds of factors through contacting with methioninase; As with this cell or contain the amount of the methioninase that culture medium contacted of this cell; The amount of methionine; The specific activity of enzyme, temperature influence the reaction condition of response speed with other and the parameter that is easy to control by the implementer etc.Be about 10 minutes to 30 days typical period, preferred about 1 hour to 20 days, and more preferably about 1 to 10 day.

The stagnation of cell cycle is a kind of like this state, and wherein cell neither divides also and do not circulate through whole periodic processes, and each stage is called G respectively ₀, G ₁, G ₂With the S phase.As confirming that through accumulation in case remove methionine according to thinking, cell cycle can stop at S/G with respect to normal resting cell DNA ₂Stop late period.Measure through various Histological methods, method that can the identification of cell cycle arrest, and can estimate with cell culture, this method as be shown in the examples comprises the dna content of measuring cell mass.

The tumor that this Therapeutic Method can be used for comprises the malignant cell of any malignant cell type as in solid tumor or neoplastic hematologic disorder, finding.The example of solid tumor includes but not limited to the tumor of following organ: pancreas, colon, caecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate and breast.The example of neoplastic hematologic disorder comprises bone marrow tumor, T or B cell malignancies, leukemia, lymphoma, blastoma, myeloma etc.

In a scheme; A kind of compositions that can tolerate on the physiology of methioninase of the present invention that contains through intravenous or intraperitoneal injection administration patient effective dose is accomplished intravital contact, removes the methionine source of the tumor cell that is present in patient's body-internal-circulation thus.Also can be through accomplishing the contact of methioninase to methioninase to the administration of organizing that contains tumor cell.

The treatment effective dose of methioninase is to reach that the methionine in tumor tissues or the patient circulation is promptly removed in required effect and therefore the premeasuring of calculating makes tumor cell stop division.

So the dosage range of taking methioninase of the present invention is to be enough to produce the dosage range that required effect promptly reduces the disease of tumour cell division and cell cycle.This dosage can not be greatly to causing disadvantageous side effect, like hyperviscosity symptom, pulmonary edema, congestive heart failure etc.To change along with the different of patient's age, health, sex and disease degree through dosage, this is that those of ordinary skills can determine.

If there is the complication doctor can adjust this dosage.

The treatment effective dose of methioninase of the present invention generally is a kind of like this dosage; Be enough to make blood vessel interior (blood plasma) or partial methioninase concentration to reach every milliliter of about 0.001 to 100 unit (U) when promptly taking the compositions that can tolerate on a kind of physiology; Preferably more than 0.1U, more preferably every milliliter more than the 1U methioninase.Common dosage is to be as the criterion with body weight to take, and at approximately 5-1000U/kg/ days, preferably approximately 5-100U/kg/ days, more preferably about 10-50U/kg/ days, most preferably in about 20-40U/kg/ days the scope.

In a preferred method, used methioninase is substantially free of endotoxin, further discusses as this paper.Particularly preferably be and use the endotoxic methioninase that is substantially free of that recombinant produced.

This methioninase can through the injection or gradually infusion a period of time through the intestinal external administration.Methioninase also can pass through intravenous, intra-arterial, intraperitoneal, trans-oral, intramuscular, subcutaneous, intracavity, percutaneous, percutaneous drug delivery; Can transport through the wriggling mode, perhaps be injected directly into the tissue that contains tumor cell or come administration through the pump that links to each other with the conduit that contains potential biosensor or methionine.

The common intravenous administration of therapeutic combination that contains methioninase is like injection units dosage.Term " UD " is meant the physically discrete unit of the single dose that is suitable for the curee when being used for the description of therapeutic combination of the present invention, active substance and required diluent that each unit contains the premeasuring that can produce required therapeutical effect are carrier or excipient (vehicle).

Said composition is to treat the effective dose administration with the mode consistent with dosage form and a kind of.The amount of being given depends on the object of being treated, and this objective system is to using the ability and the effective degree of required treatment of active component.Need the accurate amount of the active component of administration to depend on doctor's judgement and all be that each individuality all is unique.But, herein disclosed is the suitable dosage range that system uses, it depends on the approach of administration.Also thought over the method for suitable initial administration and booster dose injection, the characteristic of this method is that initial administration is then injected again or the multiple dosage of administration at the interval of one or more hour.This paper has described the example of multiple dosing, and it especially preferably continues to keep serum or organizes the high-load of methioninase and continue the low content keeping serum or organize methionine on the contrary.On the other hand, also thought over the continuous intravenous injection that is enough to be maintained to the concentration in the intravital blood special range of interior therapeutic.

II. The removal method of methionine

In realizing method of the present invention, through can be the material that contains methionine also can be with removal methionine that above-described methioninase contacts the time with one or more additional methionine removal steps.And the present invention has considered to remove the various treatment steps of methionine, and this paper can further describe.

A. The methionine starvation method

For example; Can use the hungry step of methionine to come to reduce first methionine content on every side; Wherein should the hunger step comprise that contacting a period of time to tumor cell with the nutrient that does not contain methionine was the methionine hunger period, this step can carried out with the methioninase contact procedure before or in the process.The culture medium that the hungry step of methionine can be included in external use shortage methionine is low content or the culture medium that does not have methionine, and perhaps the interior use of body does not contain the diet of methionine.The culture medium that does not contain methionine is known in field of tissue culture.An example of this culture medium is the minimal medium (lacking methionine and choline chloride) that contains the Eagle of non essential amino acid, as obtaining from GIBCO.The aminoacid diet that lacks methionine also can obtain with commercial sources, comprises from Teklad, TD 92077 diet that Inc. obtains.

The time of the hungry step of methionine can change according to the difference of concrete application on a large scale, and it is to be enough to make cell culture, and the methionine lowering of concentration of tissue or blood vessel is to reduced levels and stop further time of decline of this level.The typical time can be from about 6 hours to 2 months, and preferably approximately 1 day to 2 weeks.

Because methionine is a kind of essential amino acid, should be understood that in the use of many inventive method all to a certain extent normal cell is had toxicity.But, as described herein, the precursor homocysteine that normal cell and tumor cell can different ground metabolism methionine, homocysteine can replenish the shortage of methionine in the normal cell like this, does not save the dependency of tumor cell to methionine simultaneously again.

So; In a relevant scheme; The present invention has considered a kind of method of using the nutrient (culture medium or diet) that lacks methionine, and it can add precursor such as homocysteine or its analog of the used methionine of normal cell, and essential nutritional supplementation is provided.With about 5 to 200 μ M preferably approximately the concentration of 10 to 100 μ M in Nutrient medium, add homocysteine.

The precursor that is used for the preferred methionine of this scheme comprises L-homocysteine-thiolactone, homocysteine and 4-methyl sulfur-2-oxy butyrate.

So, in a scheme, the present invention includes and feed the step of mammal a period of time with the diet of methionine deficiency and remove methionine in the blood vessel.This diet can be chosen wantonly and comprise the fill-in of methionine precursor as methionine.On the other hand, this methionine precursor also can be taken through injection or other approach of knowing.As the homocysteine of diet supplement preferred every day of dosage be every day every kg body weight about 5 to about 1000mg homocysteine.

The route of administration of homocysteine is generally the same with the approach that adds methioninase, and it depends on the target tissue that transports this fill-in arrival.

B. The competitive inhibitor of methionine ester

According to the observation described in the embodiment, we further find to use the competitive inhibitor of methioninase to can be used to the effect that concertedness strengthens methionine removal method as herein described.So the present invention considers the hungry step of use methionine and comprises that also contacting one section to tumor cell with the competitive inhibitor of a certain amount of methionine is enough to and can utilizes enzyme to suppress the metabolic time of methionine with methionine.Be used to instruct the methioninase of competitive inhibitor to comprise S-ademetionine decarboxylase, methionine t-RNA synzyme and methionine adenosyltransferase.

Suppress the required time of methioninase preferably at methionine hunger period itself.

The competitive inhibitor of methionine and methionine competition utilize the substrate of enzyme as methionine, and pass through the effect of the issuable homergy of the endogenous methionine of direct competitive, that is to say the metabolism that suppresses methionine.Need the place of methionine and methionine cometabolism, this competitive inhibitor to play at cell and reduce the metabolic effect of methionine, thereby need higher methionine concentration to produce the identical effect of observed effect when not having inhibitor.

The competitive inhibitor of methionine can be any methionine derivant with typical competitive inhibitor function.General competitive inhibitor comprises the alkyl derivative (being the alkyl thionine) of methionine; Wherein the methyl of methionine (being methionine) can be replaced (ethionine) by ethyl; Replaced (rosickyite propylhomoserin) by propyl group, replaced (fourth methyllanthionine), perhaps replaced (penta methyllanthionine) by amyl group by butyl.

Consider that also a cycloleucine and halo methionine are as available methionine competitive inhibitor.General halo methionine is selected from fluoro methionine, chloro methionine, bromo methionine and iodo methionine.So the methionine competitive inhibitor of being considered is selected from some materials that comprise alkyl thionine, cycloleucine and halo methionine, wherein said alkyl thionine is not a methionine.

Amount to the effective methionine competitive inhibitor of competitive inhibition methionine is the amount that in the valid density of methionine, can produce the effect of reduction.This amount generally is to surpass one mole with respect to the methionine that exists, and this is as being shown among the embodiment.The scope that suppresses dosage generally is with respect to methionine concentration in the culture medium that has the methionine of being competed, and surpasses 10 to 1000 times of moles, preferably surpasses at least 20 times of moles, more preferably surpasses at least 50 times of moles.As shown here, when using inhibitor in vivo, dosage generally is every kilogram of about 5-30mg of the weight of animals, preferably approximately 25mg/kg.

The amount of required inhibitor can depend on the amount of the endogenous methionine that exists when taking this inhibitor in the time of effectively.Can confirm the relation between the valid density of methionine concentration and inhibitor through the dose titration result who is shown in an embodiment, and can limit through the type reaction speed theory of competitive inhibitor.The typical amounts of inhibitor is to about 1mM from about 10 μ M.

And; Can predict the effective dose of inhibitor through vitro tissue cultural method described herein; This method comprises and at first adopts any method described herein in removing the specific tumor tissue of methionine, to set up effective condition, then measures the valid density of inhibitor.

Use various indicators to comprise that the indicator described in the embodiment can monitor the metabolism of using the methionine competitive inhibitor to suppress methionine.These indicators are included in fed in the mice that does not contain the methionine diet, measured the dna content of tissue culture, stopped and strengthening the indicator that suppresses tumor tissue growth as cell cycle.Other indicators also are very clearly concerning those of ordinary skills.

C. Use multiple methionine to remove the synergism of method-anti-methionine amic therapy method

In a scheme, the synergism that takes place when using two or more different methionine to remove method in order to be utilized in, the present invention considers that use often is called the multiple methionine removal method of anti-methionine amic therapy method.

Methionine removal method described herein comprises that (1) use does not contain the culture medium (external) of methionine or the methionine starvation method of diet (in the body); (2) be exposed to methioninase and remove the endogenous methionine with the method that adopts enzyme; (3) reduce the valid density of endogenous methionine with the methionine competitive inhibitor; Particularly with the combining of above-mentioned (1) and (2) method; Wherein the content of methionine reduces, and (4) use the precursor and the homocysteine described herein of methionine to increase any selectivity to tumor cell in other three kinds of methionine removal methods.

So, concerning the removal of methionine, can use any combination of above-mentioned defined method; Comprise (1)+(2); (1)+(3), (2)+(3), any adding (4) of (1)+(2)+(3) and these four kinds of combinations, carried out the chemotherapy of anti-methionine described herein.

Concerning removing methionine and its metabolite to greatest extent, preferred especially use does not contain methionine but contains competitive inhibitor and the methioninase that methionine precursor also uses methionine simultaneously.

III. the oncotherapy that strengthens antimitotic agent acts in another scheme; The present invention considers in method and to use methionine removal method as herein described to increase (enhancing) routinize selectivity of the used antimitotic drug of the effectiveness and the selectivity, particularly treatment of cancer of therapy.

Methionine remove main purpose that step and conventional resisting mitosis therapy be used in combination be utilize such a case promptly before mitosis methionine remove method can be optionally and stop the hypertrophy of tumor cell specifically; Like this in case use again the ample supply methionine to give cell, tissue, culture medium or vascular system; Immobilized cell can begin cell cycle synchronously, makes the consistent hypertrophy of cell mass and receive the chemotherapeutic effect of cell cycle specificity easily.In order to make tumor cell get into the cell lag phase, can use the cytotoxic agent of any cell cycle.This medicament is known those of ordinary skills.In a preferred scheme, the cytotoxic agent of this cell cycle is a kind of antimitotic agent.

So in a preferred scheme, the present invention considers a kind of anti-tumor chemotherapeutic method, it comprises that removing step with methionine as herein described forms immobilized tumor cell, then with other step:

(a) contact the mitosis that starts immobilized tumor cell to immobilized tumor cell with the methionine that brings out the cell cycle consumption, form mitotic cell and

(b) contact mitotic cell with a certain amount of antimitotic agent, this consumption is enough to suppress the mitosis of mitotic cell, suppresses the growth of tumor cell thus.

Be called the cell cycle inducing agent to methionine, methionine salt and function equivalent thereof with the cell cycle function that starts akinete, and can use together or on the other hand in the tumor cell group of having removed methionine as the cell cycle and the mitotic reagent that start in one or more akinetes.

The amount of bringing out cell cycle of cell cycle inducing agent is in the tumor cell group of having removed methionine, to start (10%) at least, preferred great majority, the more preferably consumption of the cell cycle at least 90% the akinete.Using-system is learned or metabolic label can be measured the startup and the degree of cell cycle at an easy rate.Concerning methionine, the consumption that brings out cell cycle is generally at the scope of about 1 micromole (μ M) to about 2.5 mMs (mM), preferably approximately 10 to 250 μ M.When the ample supply methionine, likewise can be used to attack still any tumor cell to the specific drug of S phase in the S phase.

The approach that contacts the cell cycle inducing agent with immobilized tumor cell (use) can change, but general and described herein to take the used approach of methioninase or competitive inhibitor identical.

The arrangement of time that immobilized tumor cell is contacted with the cell cycle inducing agent can change according to tumor and the different of other considerations with period, and this consideration can be rule of thumb through deciding like method for tissue culture described herein.The cycle inducing agent can have a separating medium almost to add simultaneously before antimitotic agent or with anti-.Specifically anti-ly have a separating medium to work rapidly and bringing out of cell cycle is slowly the time when being measured to, it is favourable before antimitotic agent, adding inducing agent.These parameters depend on the particular type of tumor, the position of tissue, the selection of antimitotic agent etc.And, using Therapeutic Method in vivo before the administration, can measure the selection of selection, dosage and the medicine of optimized variable such as time at an easy rate through the whole bag of tricks.Preferred optimization method is to use tissue culture described herein to measure the example of system.

Bringing out the cell cycle of immobilized tumor cell depended in the selection that contacts a kind of arrangement of time of antimitotic agent.The cycle whenever generally can both be antimitotic agent and mitotic cells contacting, and make cell become mitotic cell at once.Concerning some tumor cell group, this situation can take place rapidly after adding inducing agent, make can be in immobilized tumor cell simultaneously or intimate inducing agent and the antimitotic agent of adding simultaneously.On the other hand, can add antimitotic agent after about 1 hour to 30 days at the adding inducing agent, but preferred within a day or two days of bringing out.

Antimitotic agent used herein can be to hyperplasia, any reagent that silk separates or cell cycle has the basic mechanism of cytotoxicity is arranged with the special preparation of other cell cycles.So antimitotic agent can comprise that all cpds is an antimetabolite, or other have in the toxic chemical compound any one to division (mitotic) cell.The preferred clinical pharmacology that is used for the antimitotic agent of the inventive method is the inhibition that pair cell has an isolating active, the inhibition of antitumor agents such as, alkylating agent synthetic to nucleic acid, antibiotics, alkaloid.Improve constantly with regard to the pharmacology field; Should be understood that the present invention is not limited to present known antimitotic agent and other cell cycle specific preparations, but comprise that the chemical compound that uses known equivalent, new discovery or exploitation and other have necessary active reagent.

The example of alkylating agent comprises cyclophosphamide (CTX; Cyclophosphamide), chlorambucil (CHL; Dicentrine), cisplatin (CisP; CDCP), two busulfans (Busulfan), alkeran, carmustine (BCNU), streptozotocin, triethylene melamine (TEM), ametycin and other alkylating agents.

The example of antimetabolic product comprises methotrexate (MTX), etoposide (VP16; Etoposide), 6-mercaptopurine (6MP), 6-thiocquanine (6GT), cytosine arabinoside (Ara-C), 5-fluorouracil (5FU), nitrence azoles ammonia (DTIC) and other antimetabolites.

The example of antibiotics comprises unwrapping wire line rhzomorph D, amycin (DEX; Adriamycin), many promises mycin (daunorubicin), bleomycin A5, mithramycin and other antibiotics.

Alkaloidal example comprises Changchun alkaloid such as vincristine (VCR), vinblastine etc.

Other antitumor agents comprise taxol and derivant thereof, cell growth inhibiting agent glucocorticoid such as dexamethasone (DEX; Dexamethasone) and corticosteroid such as prednisone, nucleosidase inhibitor such as hydroxyurea, aminoacid are removed enzyme such as asparaginase and other panoramic antitumor agents.

Synthetic and the preparation of above-mentioned antimitotic agent (cytotoxic agent) all is that people know, and description is all arranged in various raw datas, so just no longer repeat here.Raw data example synthetic and the preparation mentioned reagent comprise " internist's desktop with reference to " ( Physicians Desk Reference), Barnhart, eds., Medical Econom ics Company, Inc., Oradell, N.J., 1992; " Merck index " ( Merck Index), the 11st edition, Merck & Co., 1989.

In embolic chemotherapy, using above-mentioned cytotoxic agent generally is the obvious characteristic of field of cancer, uses them here, also relates to monitoring toleration and effectiveness and control route of administration and dosage.For example, the actual dose of this cytotoxic agent can change according to the patient's who is cultivated who uses existing method for tissue culture to measure cell effect.General this dosage will reduce than the employed amount of the cell cycle of lack of synchronization, and this is as shown in the description of the present invention.

The typical doses of effective cell toxin agent can be in the scope that manufacturer is recommended, and can be reduced to the concentration or the consumption of an about at the most magnitude by vitro reactions or the represented dosage of animal model reaction.So; This actual dosage will depend on doctor's judgement; The effectiveness of patient's body situation and Therapeutic Method, this effectiveness with the vitro responses property of the tissue sample of the malignant cell of initial cultivation or tissue culture or in appropriate animal model viewed reaction be as the criterion.

IV. Gene therapy

In another scheme, according to the contact method of methioninase of the present invention, the present invention considers at tissue or in by the lymphocyte around the treated tissue and uses rDNA to express methioninase.For obtaining this result, can expect through the expression of controllable methioninase expressing gene and thus, the methioninase gene outcome of generation and be used for contact procedure methioninase is provided.

As described above, at present known have a large amount of suitable expression vectors to can be used to express the methioninase that can use.In a scheme, the methioninase expression of gene is controllable.So an expression vector has the second nucleotide sequence of article one sequence that can be operably connected to the coding methioninase, this has also defined the means of regulating methioninase gene expression.An example of this regulating measure is an inducible promoter, and most preferred promoter is a tumour-specific.With the expression model of a kind of promoter of tumour-specific control methioninase gene expression, these express models is useful especially to gene therapy as herein described.

The one type of promoter that can bring out responds to the component that can add in the culture medium, or is penetrated at an easy rate and expresses the methioninase gene in the host cell that contains gene.The promoter of other type is aforesaid tumor-specific promoters, for example carcinoembryonic antigen (CEA) promoter.

So the present invention also considers a kind of removal method of the methionine that contacts methioninase with tumor cell group, it comprises the following steps:

(i) in vivo or in vitro; Import the cell that tumor cell or importing infiltration tumor lymphatic cell or its bone marrow precursor form the methioninase gene transfection to a kind of expression vector; Wherein this expression vector has the coding methioninase and can regulate the nucleotide sequence of the expression of methioninase, and has the nucleotide sequence that the means of regulating the methioninase expression can be provided;

(ii) the cells contacting of intravital tumor cell and methioninase gene transfection; With

(iii) in cells transfected, express the nucleotide sequence of coding methioninase, in this cells transfected, produce methioninase thus and contact the methioninase that is produced with tumor cell.Concerning those of ordinary skills " importing " be meant any known method of inserting expression vector target cell in one way, wherein this target cell can be expressed genes carried in this expression vector.This insertion can be carried out in vivo or in vitro.Can accomplish this " importing " through the method for for example transfection, conversion or viral infection.

No matter use various known transfection methods can realize tumor cell, be in adherent cell or the suspension or the infection of intravital cell.After this, import cells transfected (if using sv transfection method) again the host and make this transfectional cell be positioned at natural, near relevant, the tissue that will treat, contact cells transfected with the tumor cell that will treat thus.Use various means can realize importing cells transfected to methioninase, exist but generally need selected marker.

In a scheme, the instrument that regulator gene is expressed is the promoter of bringing out that the reagent that can add in the culture medium is replied.Promoter like metallothionein.On the other hand, the promoter that can bring out can be a tumor-specific promoters.This promoter works to the expression of the target methioninase of tumor cell.

Wanting cells transfected can be that tumor cell sample or normal immunocyte are as soaking into the cell of tumor, as soaking into tumor lymphatic cell or the bone marrow precursor of bone marrow such as the stem cell or the germinal cell of hemopoietic.These cells can be in vivo or are external.

Embodiment 9 has described the separation of the nucleic acid molecule of coding methioninase.This embodiment has also described and has been used for the generation that the expression vector of methioninase is produced in zymolysis in batches.Skilled those skilled in the art can use clone's methioninase gene at an easy rate, like what in pAC-1, provided, process the expression cassette that is applicable to the said gene therapy.

C. Therapeutic combination

In another scheme, the present invention considers therapeutic combination, and it contains the methioninase and the medicine acceptable carrier isolated basically, that preferably produce with the recombinant method of treatment effective dose.

L-methioninase (L-methionine-α-deaminizating-γ-sulfydryl methane-lyase or methioninase) is through deamination and desulfurization acute pyogenic infection of nails base be used for degrading a kind of enzyme of methionine.The activity of methioninase can the amount of formed α-butanone hydrochlorate record when being determined at the methionine cracking at least.The methioninase of a unit (U) is defined as under the standard test condition amount that from methionine per minute produces the enzyme of 1 mM α-butanone hydrochlorate, like Ito etc., " biochemical magazine " ( J.Biochem.), 79: 1263-1272,1976; And Soda, " biochemical analysis " ( Analyt.Biochem.), 25: 228-235, described in 1968.

Methioninase can be comprised directly and being separated by bacteria culture media by the preparation of many sources, is perhaps expressed by the recombinant DNA molecules of encoding proteins acid pheron, for example is described in example 9 and example 12.

The bacterial origin of methioninase comprises pseudomonas putida (Pseudomona Dutida), the potential source of ovum shape pseudomonas (Pseudomonas ovalis) and Aeromonas Pseudomonas (Aeromonas) and any other methioninase.The pseudomonasputida bacterial strain can commercial sources obtain from ATCC, and go into hiding of it number is respectively ATCC 8209 and ATCC 7955.Other antibacterials generally also can obtain from academic research group.Described at present and made the purification methioninase that ins all sorts of ways.For example, referring to Kreis etc., " cancer research " ( Cancer Res.), 33: 1862-1865,1973; Tanaka etc., " European biochemistry association community communication " ( FEBS Letters), 66: 307-311,1976; Ito etc., " biochemical magazine " ( J.Biochem.), 79: 1263-1272,1976; Nakayama etc., " agricultural biochemistry " ( Agric.Biol.Chem.), 48: 2367-2369,1984 and Soda, " biochemical analysis " ( Analyt.Biochem.), 25: 228-235,1968.But these methods all do not obtain the highly purified endotoxic methioninase that do not contain.The invention provides two kinds of methods that are used for the purification methioninase, this methioninase contains endotoxin hardly like this.

A kind of preferred methioninase has the specific activity of every milligram of about 10 to 50 units of albumen (U).This paper has described the exemplary formulations of purification methioninase, and said preparation has about 10 to about 50U/mg specific activity, in embodiment 12, uses the prepared methioninase of expression vector pAC-1 to have the specific activity of about 20.1U/mg.

A kind of preferred methioninase is preferably isolating substantially.Separate being meant this enzyme substantially, preferred at least 90% purity, more preferably at least 99% purity or be uniform basically by weight in respect of at least 50% purity.When in electrophoretic medium such as PAGE (PAGE), analyzing, preferred albumen is uniform basically.Evenly being meant on PAGE has only single mensuration band.

Owing to when contacting, do not hope that the side effect relevant with endotoxin arranged, so preferably methioninase is to be substantially free of endotoxin such as bacillary lipopolysaccharide through intravenous injection or intraperitoneal administration and with mammal physiology.Being substantially free of here is meant that every milligram of (mg) methioninase albumen to being less than about 10ng endotoxin, preferably is less than 1ng endotoxin/mg methioninase, the preferred 0.1ng endotoxin/mg methioninase that is less than.Measuring endotoxin is known in the treatment field, and making ins all sorts of ways can carry out measuring endotoxin.Preferable methods has been described in an embodiment.

Preferred methioninase is heat-staple, when using under the condition of elevated temperature in like animal body, can increase its storage period and effectiveness like this.Thermally-stabilised this endonuclease capable that is meant descends to expose 10 minutes and keeps its specific activity to reach 80% preferably to keep 90% specific activity at 60 ℃, more preferably keeps 95% specific activity.

Preferred methioninase has at least 5 hours serum half-life, preferred 6 hours, more preferably at least 7 hours.

Preferred especially methioninase is to prepare from the expression vector pseudomonas putida preparation or that use the dna molecular of from pseudomonas putida, separating that contains the methioninase of encoding.The pseudomonas putida methioninase has the apparent molecular weight of about 43 kilodaltons when PAGE-SDS analyzes under the degeneration condition.The method for optimizing of purification methioninase has been described in an embodiment.

So therapeutic combination contains the carrier that can tolerate on a kind of physiology and is dissolved in or is dispersed in the isolating substantially methioninase as active component wherein.In a preferred scheme, when dosing a patient with for therapeutic purposes, these therapeutic combination right and wrong are immunogenic.

The characteristic of a scheme of the present invention is the methioninase of chemical modification, and it comprises the methioninase that is attached on the polymer.Preferred this methioninase is isolating substantially and contains endotoxin hardly." chemical modification " is meant to be changed and forms any type of methioninase different with the methioninase of purification from nature.Preferably come the chemical modification methioninase through being connected to this methioninase method on polymer such as the Polyethylene Glycol.

As used herein; The term " pharmacy is acceptable ", " physiology can tolerate " and they that are used to refer to component, carrier, diluent and reagent can exchange use in phraseological variation, and they all represent can this material administration mammal or the mankind take or produce undesirable physiological action like nauseating, dizzy and stomach upset etc. taking Shi Buhui for the mammal or the mankind.

In the art to comprising that the preparation of drug combination that is dissolved in or is dispersed in active component wherein is very clearly.Generally can process injectable aseptic liquid solution such as suspension, moisture or water-free solution to this compositions, perhaps also can be made into the solid form that is applicable to solution and suspension of liquid before use.Said preparation also can be the Emulsion form.Phospholipid and the liposome composition of particularly preferably being as described herein.The methioninase that in addition, on ointment or dispersibility cover plate such as binder, also can contain therapeutic dose is to provide the part release of this reagent.

This active component can be with medicine acceptable and with the compatible mixed with excipients of active component, its consumption should be suitable for Therapeutic Method as herein described.Appropriate excipients for example has water, saline, glucose, glycerol etc. and compositions thereof.In addition, if desired, said composition can contain auxiliary substance such as wetting agent, emulsifying agent, the pH buffer agent of the effect that can improve this active component of trace etc.

Therapeutic combination of the present invention can comprise the acceptable salt of the medicine of component.The acceptable salt of medicine comprises the acid-addition salts (formed with the free amine group of polypeptide) that perhaps resembles organic acid formation such as acetic acid, tartaric acid, mandelic acid with the mineral acid of example hydrochloric acid or phosphoric acid.With the formed salt of free carbonyl also can for example hydroxide and organic base such as 2-aminopropane., front three ammonia, 2-ethylaminoethanol, histidine, the procaine etc. of sodium, potassium, ammonia, calcium or ferrum be derived by inorganic base.

The carrier that can tolerate physiology in this area is known.The example of liquid-carrier has the aseptic aqueous solution that except that active component and water, does not contain other materials, or contains sodium phosphate, the normal saline of buffer such as physiology pH value or the two is like phosphate-buffered saline.Aqueous carrier also comprises more than one buffer salt and resembles sodium and the chloride of potassium, glucose, propylene glycol, Polyethylene Glycol and other solutes in addition.

Fluid composition as described herein also can comprise the liquid phase of dissolving with water and with water-insoluble liquid phase.This additional liquid phase example is glycerol, vegetable oil such as Oleum Gossypii semen, organic ester such as ethyl oleate and oil-in-water emulsion, especially foregoing liposome composition.

This therapeutic combination contains the methioninase of effective dose, and general effective dose is the activated protein of every therapeutic combination gross weight at least 0.1 percentage by weight, and preferably about at least 25 percentage by weights.Percentage by weight is the weight ratio of methioninase and total compsn.So for example 0.1 percentage by weight is the methioninase of per 100 gram total compsns, 0.1 gram.

Can use in vivo through blood vessel in order to make the methioninase compositions; We consider the scheme of therapeutic combination of a kind of sustained release methioninase of preparation, and optionally methioninase albumen is not degraded and can reduce the influence of other phenomenons of serum half-life of the methioninase of treatment administration.

So; In a scheme; The present invention considers and comprises the therapeutic combination that transports carrier that this transports carrier and comprises carriers such as polymer, polymer support, granular substance, emulsion, aggregate, ion exchange resin, liposome, enteric coating, medium, biological adhesive, microcapsule, hydrogel.Comprise liposome drug release carrier example at least by Tarcha at " polymer of control drug release " (" Polymers For Controlled Drug Delivery "), CRC publishing house, Boca Raton described in 1990.

The present invention also provides the detailed method of purification methioninase, compares the known method of this method and other, and it can provide superior productive rate and purity.Purification of Recombinant methioninase, special purification are to use the method for optimizing of the methioninase of the conversion host generation that efficiently expresses module that contains the coding methioninase, comprise the following steps:

A) in aqueous buffer solution, heat the extract of the transformant that contains methioninase about 1-10 minute down in about 40-60 ℃, preferably 50 ℃ of heating 1 minute.

B) in the GS-3 rotor (Sorvall, Du Pont) with about 10k to the extract centrifugalize about 15 minute to 1 hour of 20k rpm rotating speed after, preferably 4 ℃ of about 13k rpm centrifugalize 30 minutes heating;

C) use 10mM kaliumphosphate buffer (pH8.3), use the filter ultrafiltration supernatant liquid of about 50k, be preferably Millipore Pre/Scale:TFF PLHK 100K 2.5ft to the 100k aperture ²Filter cartridge;

D) at LIS (approximately 10-50mM) KCl and under the condition of the about pH7.0-7.6 of 10-20mM kaliumphosphate buffer, carry out the DEAE ion-exchange chromatography; And collect the fraction that contains methioninase with the KCl gradient solution eluting of 40-200mM, preferably use the DEAE-SepharoseFF post;

E) at ionic medium intensity (approximately 50-100mM) KCl and under the condition of the about pH8.0-8.6 of 10-20mM kaliumphosphate buffer, carry out the DEAE ion exchange chromatography chromatography second time; And collect with the KCl eluting of 100-200mM and with the fraction that contains methioninase of phosphate buffer (pH8.3) eluting, preferably use DEAE-agarose FF post; With

F) the fraction of in step (e), collecting with can adsorb endotoxic column chromatography medium and contact; And collection eluent; From this eluent, remove endotoxin formation thus and do not contain endotoxic methioninase; This methioninase has the endotoxin that the active and every milligram of albumen of the methioninase of at least 20 units of every milligram of albumen has about 1-100ng, preferred

post that uses.

This cell extract preferably makes from the reformed host cell that can express high-load RMETASE (approximately 5-75% total cell protein).On the other hand, also can be the Organic substance of natural expression methioninase as starting material.Concerning the bacterial cell extract; The preparation extract generally is at first to gather in the crops and wash the bacterial cell culture to form cell paste/ball; And depending on that whether passing through centrifugal separation method still is hollow fibre pipe filter method results, these methods generally are called optical imaging.

Then, use conventional instrument cracked cell.Preferred homogenizer such as cavitation type homogenizer (Microfluidics Corp.Model#HC 8000 fragmented cells for example that use.

The albumen and other insoluble matters of resulting suspension heating with the deposition selection.Heating condition generally be about 45-60 ℃ following 1-10 minute.Preferred 50 ℃ of heating stepses of 1 minute.

Remove fragment to the extract centrifugalize after the heating, and filtering supernatant is used further to the DEAE ion-exchange chromatography medium in above-mentioned two steps.Preferred absorption and elution requirement have been described in an embodiment.In these steps, can use any of various DEAE ion-exchange chromatography media, and the selection of medium is not limited.Commercial source comprises Pharmacia Fine Chemicals, BioRad and Sigma.

Afterwards, remove endotoxin production and have the endotoxic albumen of accepting content as foregoing.Can both carry out endotoxic removal step with any in the various tool of knowing; This removal step generally comprises and contacts the albumen in the solution with adsorbing endotoxic a kind of column chromatography medium, obtains containing the column chromatography medium eluate that does not contain endotoxin protein.Being used to remove endotoxic preferred commercial reagents is

D. The methioninase of chemical modifying

For half-life and immunogenicity that reduces it or the antigenicity that prolongs methioninase, can combine methioninase with a kind of polymer.

In oversensitive individuality, there is allergic probability.On the other hand, the antibody of anti-methionine enzyme can shorten the half-life of methioninase.

Attempt various approach at present and solved polypeptide and proteic antigenicity problem.Is polymer such as polyethylene glycol conjugation one of approach that reduces protein antigenicity to albumen.The present invention relates to use the chemical modification of the methioninase that a kind of novel and effective method produces, it comprises in following the purification methioninase of condition and Polyethylene Glycol (PEG) coupling that keep enzymatic activity.The half-life that PEG-methioninase of the present invention has prolongation does not have apparent immunogenicity.

New has half-life and the reduced immunogenicity that high methionine is removed activity, prolonged with the bonded methioninase of Polyethylene Glycol (" PEG-methioninase " or " methioninase of PEGization ").Therefore the PEG-methioninase provides a kind of maintenance stability, prolongs serum half-life, reduces immunogenicity and the new tool that reduces toxic inhibition tumor growth.

The present invention stable, methionine removes effectively, the long half-lift of having and the immunogenic PEG-methioninase of right and wrong can be as multiple dose antitumor agent safely and effectively.The PEG-methioninase can be kept in the liquid preparation of the 0.12M sodium chloride in the 10mM sodium phosphate (pH 7.2) under-70 ℃, 4 ℃ and room temperature, and loss of activity not.

Importantly, PEG-methioninase right and wrong of the present invention are immunogenic, and this makes it be particularly suitable for treating responsive and need the cancer patient of repeat administration to methioninase.This area ordinary technical staff understands that with the same with methioninase, the PEG-methioninase can provide with multiple dosage form.

The preferred formulation that does not have endotoxic methioninase of purification is to be dissolved in the 0.12M sodium chloride solution in the 10mM sodium phosphate buffer (pH7.2) with the concentration between 0.06M and 0.2M.Activity is about 10 unit/mg.

The methioninase of PEGization can be through the known method test of this area ordinary technical staff.For example can measure pharmacology, safety and the functional character of the PEG-methioninase of the inventive method preparation with in vivo test.

These tests can comprise acute toxicity, pharmacokinetics, the removal of methionine in serum of measuring the PEG-methioninase, and the immunogenicity of evaluation PEG-methioninase.

The no endotoxic PEG-methioninase of purification is injected in the tail vein of Mus, and blood sample collection every two hours.Measure the level of methioninase through determination of activity.Measure the level of methioninase after the methionine derivatization with HPLC.

Can in any suitable experimental animals, carry out studies on acute toxicity, illustrated like embodiment 5 and 15.In the mice body, 10-100 unit/1-10mg PEG-methioninase is injected its tail vein.Record live body signal and visual observations.Blood sample collection before injection, and after injection blood sample collection every two hours.Measure the active and methionine level of methioninase.Also measure the function of kidney and liver function.With measured by standard techniques tissue there is free of toxic effects like lung, kidney, liver and brain.Also analyze blood sample and bone marrow.

In another embodiment, methioninase combines with a polymer, has produced a kind of immunogenic compositions that has basically no, and also causes the increase of active half-life of methioninase in the body.

In one example, methioninase is through combining to have carried out chemical modification with a polymer.

In another example; Methioninase is through by chemical modification, the example of polyalkylene oxide includes but not limited to PEO, PPOX, ethylene oxide copolymer and epoxy propane copolymer with the combination of polyalkylene oxide (polyalkylene oxide).

In preferred embodiment, methioninase through combining with Polyethylene Glycol by chemical modification.

In another preferred embodiment, be substantially free of endotoxin in conjunction with the methioninase of Polyethylene Glycol.

The present invention also provides a kind of pharmaceutical composition, and it contains the methioninase that combines with polymer of treating effective dose.This polymer can be a polyalkylene oxide, such as but not limited to the copolymer of PEO, PPOX, ethylene oxide copolymer and expoxy propane.

A kind of pharmaceutical composition is provided in a preferred embodiment, its contain the treatment effective dose with the bonded methioninase of Polyethylene Glycol.In another preferred embodiment, pharmaceutical composition contain the treatment effective dose be substantially free of endotoxic and with the bonded methioninase of Polyethylene Glycol.This compositions can prepare in the isolating material from the organism of natural production methioninase, perhaps preferably from being changed host's preparation of expressing methioninase.

A kind of method for preparing no endotoxin methioninase also is provided, and said method is through no endotoxin methioninase and polymer coupling, and generates the no endotoxic methioninase that has basically no the immunogenicity chemical modification.

In a preferred embodiment, this method comprises methioninase and polyethylene glycol conjugation.

In another preferred examples, through no endotoxin methioninase and butanimide carbonic acid methoxy macrogol ester reaction carrying out coupling.

A kind of method of treating tumor patient also is provided, has comprised the methioninase of administering therapeutic effective dose.

In a preferred embodiment, methioninase has basically no endotoxin.

In another preferred embodiment, methioninase combines with polymer.

In another experiment, polymer is a kind of polyalkylene oxide.

In another preferred embodiment, methioninase combines with Polyethylene Glycol.

E. The RMETASE preparation

The present invention further provides lyophilizing or crystalloid methioninase.At length say, found to use means known in the art, methioninase lyophilizing easily or crystallization.The methioninase preparation of finding gained crystallization or lyophilized form has high stability, is prone to hydrated, and keeps high activity after the rehydration.

Can obtain the methioninase of crystallization or lyophilized form with various approach well known.Use Verdis in an embodiment, freeze-drying apparatus 24 is at 100 millibars;-80 ℃; 72 hours, carry out the lyophilizing and the crystallization of methioninase, those skilled in the art can easily adopt other methods known in the art to be used to prepare the methioninase of lyophilizing or crystal form.

F. Dna fragmentation and carrier

I. the encode dna molecular of methioninase

DNA isolation molecule and promoter through the coding methioninase that is operably connected when having been found that in importing proper host cell; Particularly rna polymerase promoter is like being operatively connected of T7 RNA polymerization promoter, and RMETASE can be expressed with 5～75% level of about total cell protein matter.Express the high level expression model of high-level RMETASE when therefore the invention provides among the host under importing appropraite condition.

Here employed high level expression model, or expression model of the present invention is meant the nucleic acid molecules of the expression regulation element of transcribing and translating of the nucleotide sequence that is operably connected that comprises one or more guidance coding methioninases.Expressing model can be that isolated nucleic acid molecule perhaps may reside in (hereinafter explanation) in the carrier.

The present invention expresses model and comprises the controlling element that instructs RMETASE to produce, and makes the RMETASE of producing be equivalent to the 5-75% of about total cell protein matter, preferably more than 10% of total cell protein matter.Preferred control element rna polymerase promoter of expressing, more preferably T7 rna polymerase promoter.The additional embodiments of rna polymerase promoter includes but not limited to Tac and Trc promoter.

Promoter is the expression regulation element that is made up of the DNA sequence that allows RNA polymerase to combine and to transcribe.The promoter sequence compatible with the specific host system is well known in the art, and general in the plasmid vector that comprises one or several suitable restriction site, to provide.Representational such plasmid vector is to comprise T7 rna polymerase promoter, the plasmid vector of PT7 and PET, and it can obtain from multiple channel, as being obtained by businessman and the American Type Culture Collection.

The present invention expresses the nucleotide sequence that model further comprises the methioninase of encoding.Just as used herein, when transcribing and translation when causing having the active proteinic generation of methioninase of the nucleic acid molecules that contains this sequence, just think this nucleic acid sequence encoding methioninase.

L-methioninase (L-methionine-α-deaminize-γ sulfydryl methane-lyase or methioninase) is a kind of through deaminizing and the degrade enzyme of methionine of desulfuration methyl effect.The methioninase activity can record through the amount of measuring α-batanone acid that the methionine cracking generates at least.A unit (U) methioninase is defined in per minute under the standard test condition and produces the amount of the enzyme of 1 micromole α-alpha-ketobutyric acid root from methionine, and these standard conditions are described in Ito etc., J.Bio chem., 79:1263-1272,1976; And Soda, " biochemical analysis " (Anayt.Bio chem).25：228-235，1968。

Coding methioninase nucleotide sequence can comprise the unaltered sequence by the organism acquisition of natural production RMETASE; Can comprise that also being changed of organism acquisition by natural production methioninase comprises one or more nucleic acid or aminoacid replacement base, disappearance or the sequence that increases.

The nucleic acid molecules of coding methioninase, no matter its change or change and can both make in any organism by natural generation protein acid enzyme, the source of optimized encoding methioninase nucleic acid molecules is pseudomonas putida (Pseudomonas putida).Embodiment 9 discloses from pseudomonas putida, the separation and the sequencing of coding methioninase nucleic acid molecules.Preferably encode the in addition source of methioninase nucleic acid molecules includes but not limited to trichomonal vaginitis (Trichomonas vaginalis), Brazilian Nippostrongylus Nippostrongylus brasiliensis, and Fusobacterium (Fusobacterium SP.).

The methioninase complete encoding sequence can make in all sorts of ways and obtained by various sources, particularly those sources of preceding text citation.It never is the interior coding methioninase nucleic acid molecules that separates of organism of pseudomonas putida that the methioninase that Fig. 8 provides is convenient to nucleotide sequence greatly.

Particularly, those skilled in the art's nucleotide sequence that can easily use Fig. 8 to provide prepares oligonucleotide primers and expresses the polymerase chain reaction (PCR) of nucleic acid molecules of biology selective amplification coding methioninase from methionine being used for.The PCR primer that the preferred sequence that provides with Fig. 8 is the basis is to being: 5 '-GCCGGTCTGTGGAATAAGCT-3 ' (justice is arranged)

5 '-CCAGGGTCGACTCCAGCGCC-3 ' (antisense)

Preferred PCR degeneration/renaturation/extension cycle of using above-mentioned PCR primer is following: 95 ℃ of first time 95 ℃ of degeneration 10 minutes; Then 94 ℃ of degeneration in 30 seconds; 60 ℃ annealing 30 seconds, carried out 5 circulations in 2 hours at 72 ℃ of extensions, 94 ℃ 30 seconds; In 25 cycles of 60 ℃ of degeneration in 30 seconds, extended 1.5 minutes at 72 ℃ then; 72 ℃ of extensions 10 minutes, carry out 25 circulations then.Pcr amplification product is two district's bands, collects 1365bp district band wherein, the ONCase-1DNA that purification becomes to insert.

Perhaps, Fig. 8 nucleotide sequence fragment can use prior art by the DNA except that the methioninase of being encoded by the separation of the organism the pseudomonasputida as probe.Contain about 18-20 nucleotide (about 6-7 the amino acid whose one section sequence of encoding) oligomer with approach well known preparation and use, and be used to detect enough strict to reduce the genomic DNA storehouse that obtains hybridization under the false positive condition.(referring to Sambrook etc., " molecular cloning " (Molecular Cloing), Cold Spring Harbor Press1989).

Can easily synthesize as the dna fragmentation (being synthetic oligonucleotide) of the Auele Specific Primer of probe or polymer enzyme chain reaction (PCR) and the gene order of coding methioninase through chemical technology; For example; The method of the phosphotriester of Matteucci etc. (" U.S. chemical institute magazine " J.Am.Chem.Soc.103:3185-3191,1981) or use automatic synthesis method.In addition, the larger dna fragment can be easily through the known method preparation, and the for example synthetic one group of oligonucleotide that limits dna fragmentation carries out hybridization and connect oligonucleotide constituting complete fragment then.

Except the method that is the basis with PCR and dna probe, the dna molecular that polyclonal antiserum that excites with the fragments of peptides that is expected to be immunogenic Fig. 8 or monoclonal anti physical ability are separated the coding methioninase.Such antibody can be used for surveying the expression library that is produced by particular organisms, like λ gtll storehouse, only if to obtain the dna molecular of coding methioninase from pseudomonas putida organism in addition.

In case obtain the nucleic acid molecules of abiogenous coding methioninase; Those skilled in the art can easily use at random or the site-specific mutagenesis method changes the methioninase coded sequence; Since improve expression; Perhaps replace, increase or lack one or more aminoacid from the methioninase of having encoded.

In an example, in given host cell, changed the expression of methioninase coded sequence, and do not changed the amino acid sequence of the methioninase that is encoded with the raising RMETASE.The expression that RMETASE improves in the specific host can obtain through one or several codon that change is present in the nucleic acid molecules, and the result is that the codon that obtains is often by host living beings be used for the encoding codon of specific methionine.The change nucleotide sequence makes it comprise preferred codon and can realize with approach well known, like direct mutagenesis or the synthetic nucleic acid molecules that comprises preferred codon.

Except that the change that influence is expressed, the nucleic acid molecules that also can change the coding methioninase is so that the gained protein purification.For example, as disclosed among the embodiment,,, can use the fused protein of Ni++ agarose gel purification gained to increase by one section polyhistidyl sequence through changing the amino or the carboxyl terminal of RMETASE.

Also can change the methioninase coded sequence and make in the amino acid sequence of methioninase that is encoded and cause variation,, replace, or lack one or several amino acid residue as increasing.The gained RMETASE preferably has the variation that causes RMETASE to have better biology or physiological property, like the activity that improves, and the Km of reduction, the immunogenicity of reduction, or the serum half-life that prolongs.Reformed like this type can appropriate design or generation at random.

When variation be the physiological property with the amino acid sequence of initial sum product albumen matter and expectation be the basis specific selection the time, said change is an appropriate design.For example the change of one type appropriate design is to replace hydrophobic amino acid to improve dissolubility with less hydrophobic residue.The method for optimizing that produces the appropriate design change is a direct mutagenesis of using mismatch PCR primer extension method.

When change was not choose reasonable, change was exactly to take place at random.Random mutagenesis technology, like chemomorphosis, PCR reorganization and linker scanning mutagenesis produce a large amount of various at random and unspecific variations in given protein coding sequence.Such method can be used for fundamentally changing the nucleic acid molecules of coding methioninase.

The form that the RMETASE that produces in such a way then changes is used for according to the whole bag of tricks screening desirable properties known in the art.The selection method of selecting to use depends on the method for mutagenesis of host, carrier and use and character to be selected.

The present invention further provides and has comprised the carrier that one or several the present invention expresses model.Carrier be can be in the host dna molecular of self-replicating.Carrier can comprise by the episomal replication starting point that has plasmid derivative naturally, and the genome duplication starting point perhaps can be derived from viral genome.The present invention expresses the selection of the carrier that model inserts such as well known in the art, directly depends on the functional characteristic of expectation, for example protein expression and want transformed host cells.

In one embodiment, carrier comprises a protokaryon replicon.Protokaryon replicon such as ColE1 replicon are well known in the art and easily are used in the present invention and express in the combination of components.In addition, but carrier can comprise coding selected marker such as drug-fast gene.

Carrier for expression of eukaryon also can be used in the present invention and express in the combination of components.Eukaryotic expression vector is well known in the art and can obtains from some commercial undertakings.Representational such carrier is PSVL and pKSV-10 (Pharmacia), and pBPV-1/pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, #31255), the carrier pCDM8 of this paper explanation and similar carrier for expression of eukaryon.The high level expression carrier can further produce with the insect cell expression system, as with the baculovirus being the expression system on basis.

Generally speaking, the generation of coding methioninase high expressed assembly typically comprises following content:

At first obtain the DNA of coding methioninase.If the sequence from bacterial origin of expectation is not interrupted by intron, then it is suitable in any host, expressing.This sequence can change over the form cutting and reclaim of being easy to through insert the sequence comprise one or several restriction endonuclease sites at methioninase coded sequence flanking region.

Then, be cut or reclaim coded sequence and have being operatively connected of high expressed control element, preferably place reproducible expression vector.Transform suitable hosts with expressing assembly or carrier then, and under the condition that realizes RMETASE production, cultivated by the conversion host.At random RMETASE is separated from culture medium or from cell.Recovery of protein and purification are not necessarily necessaryly in some instances, wherein can allow some impurity to exist.

Each step in the top step can carry out in every way.For example can obtain desired coded sequence and directly be used for suitable hosts from genomic fragment.The structure of exercisable expression vector is to use two or more suitable replicons and control element to carry out among the various hosts.If can get usually, suitable restriction site can be added to the terminal of coded sequence, and a cut gene that is inserted in these carriers is provided like this.

II. transformed host cell is expressed high-level RMETASE

The present invention further provides with the present invention and expresses assembly or carrier transformed host cells, thereby produces the 5-75% RMETASE of about total cell protein matter, preferably accounts for the about more than 10% of total cell protein quality.Host cell can be protokaryon or eucaryon host.

Any prokaryotic hosts can be used for expressing the present invention's high level methioninase encoding pack.Preferred prokaryotic hosts is escherichia coli (E.coli).Among the embodiment below, used colibacillary DH5 α and BL21 (DE 3) bacterial strain.

Preferred eukaryotic host cell comprises insect cell, yeast cells and mammalian cell, and preferred insect cell such as SP6 and vertebrate cells are like those cells from mice, rat, monkey or human fibroblast cell line.Other preferred eukaryotic host cell comprises Chinese hamster ovary (CHO) the cell CCL61 that obtains from ATCC, from the NIH Switzerland Mus embryonic cell NIH/3T3 (CRL 1658) that ATCC obtains, and baby hamster kidney cell (BHK) and similar eucaryon tissue culture cells system.

The known method of the host that uses and bearer type is realized with the conversion of high level expression reconstitution component of the present invention to suitable host through depending on usually.For the conversion of prokaryotic host cell, preferably host cell is carried out electroporation or salt processing method, for example referring to Cohen etc., Proc.Natl.Acad.Sci.USA 69:2110,1972; With Maniatis etc., " molecular cloning, experiment guide " (Molecular Cloning, Alaboratory Mammal), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982).

About eukaryotic conversion, preferred electroporation or use cation lipid, for example referring to Graham etc., Virol.52:456,1973; With Wigler etc., " institute of NAS periodical " be 76:1373-76 (Proc.Natl.Acad.Sci.USA), and 1979.

The success cell transformed, promptly comprising the cell that the present invention expresses assembly can differentiate with known technology.The cell of for example expressing the importing of assembly by the present invention and producing can be by the single bacterium colony of clone's preparation.Can gather in the crops, dissolve from the cell of these bacterium colonies and with the existence of rDNA among their DNA of known method inspection, said method is by Southern, " molecular biosciences magazine " (J.Mol.Biol); 98:503,1975, or Berent etc.; " biotechnology " be 3:208 (Biotech.), and 1985 describe.Yet illustrated as hereinafter, the present invention further provides the rapid screening method of differentiating the transformant of expressing high-level RMETASE.

III. identify the host who expresses high-level RMETASE

The present invention further provides evaluation can produce the transformed host cell of RMETASE with the level of 5-75% total cell protein matter.Particularly, find that the transformed host cell that total cell protein matter with about 5-75% is expressed as RMETASE has tangible observable pink.When obvious especially during as the host with escherichia coli.

For identifying the transformed host cell of expressing high-level RMETASE, let transformant under the condition that recombinant protein is expressed, grow on culture medium or in the culture medium, and the cell of visual inspection growth.Detect and selection and auxocyte or bacterium colony based on peach demonstration.

For using the inventive method to select, make the growth of growth transformed host cell, can use many cultivation/growth conditionss.Thereby the composition of growth medium is decided by the nutritional need of used host/vector and is used to identify the peach detection system relevant with the RMETASE high level expression and can separates the high level expression clone.Preferred culture medium is that transformed host cell can tile above that, and with the solid medium of independent colony growth, each single bacterium colony is all among the single host.Calibrating high level expression clone's method for optimizing is a range estimation growth bacterium colony.

IV. produce RMETASE with the high level expression assembly

The present invention further provides the method for producing RMETASE.Particularly, using can be with the industrial mass production RMETASE with one or several high level expression assembly host transformed of the present invention.This horizontal expression RMETASE of host that transformed with about 5-75% total cell protein matter.Use host of the present invention, those skilled in the art can easily be used for the RMETASE of various diagnosis and Therapeutic Method with means known in the art production.

Purification is with the RMETASE of the conversion host production of the high level expression model that comprises the methioninase of encoding, or the method for optimizing of the RMETASE of the host of purification natural production methioninase production comprises following step:

A) will contain the water-containing buffering liquid of the transformant extract of methioninase, in about 40-60 ℃ of heating about 1-10 minute, preferred 50 ℃ of heating 1 minute;

B) the GS-3 centrifuge rotor (Sorvall Dvpont) goes up with about 10k to 20k rpm centrifugal about 15 minutes to 1 hour of the extract of heating, preferably 4 ℃ with about 13k rpm centrifugal about 30 minutes;

C) filter that uses about 50k to 100k aperture is preferably used the Millipore Pre/Scale:TFF PLHK 100K 2.5ft of 10mM kaliumphosphate buffer (pH8.3) with the supernatant ultrafiltration ²Cylinder;

D) carry out the DEAE ion-exchange chromatography; With the LIS in about pH7.0-7.610-20mM kaliumphosphate buffer (approximately 10-50mM) KCl; Collection contains the fraction of the methioninase of 40-200mM KCl gradient elution, preferably uses DEAE-agarose FF post;

E) carry out the DEAE ion-exchange chromatography second time among ionic medium intensity (50-100mM) KCl in about pH8.0-8.6 10-20mM kaliumphosphate buffer, and collect the fraction of the methioninase that contains useful phosphate buffer (pH 8.3), 100-200mM KCl eluting.The preferred DEAE-agarose FF post that uses;

F) with the said fraction of collecting in the step (e) with can adsorb endotoxic column chromatography substrate and contact; And collection eluent; Thereby from e) removed endotoxin the said eluate of step; Generated every milligram of protein and had that at least 20 unit methioninases are active only to contain the endotoxic no endotoxin methioninase of 1-100ng with every mg protein, preferred use post.

Cell extract is preferably from being changed the host cell preparation of expressing high-level RMETASE (approximately 5-75% total cell protein matter).For the bacteriocyte extract, extract generally prepares through at first gathering in the crops and wash bacterial cell culture cellulation group/pill (depend on that results are to filter with centrifugal action or with doughnut, two kinds of methods generally are known).

Use the conventional method smudge cells then, preferably use the homogenizer smudge cells, like cavity type homogenizer, for example Microfluidics Corp.Model#HC 8000.

Heating gained suspension is with the protein and other insoluble substance of deposition selection.Typical heating condition is about 45-60 ℃ of heating 1-10 minute, and preferred steps is 50 ℃ of heating 1 minute.

The extract of centrifugal heating is removed chip, leaches supernatant, and as indicated above with two step application of samples on DEAE ion-exchange chromatography substrate.Preferred absorption and elution requirement are seen embodiment.In these steps, can use various DEAE ion-exchange chromatography substrate, choice of base is not thought qualifications.The coml source comprises Pharmacia Fine Chemicals, BioRad, and Sigma.

After this, remove endotoxin, that produces that preceding text propose has the endotoxic protein of acceptable level.The endotoxin removal step can be carried out with various known method, generally comprises the protein that makes in the solution and contacts with adsorbing endotoxic column chromatography substrate, obtains containing the column chromatography substrate eluent that has or not endotoxin protein matter.Being used to remove endotoxic preferred merchant, to sell reagent be

G. uses methioninase prevention and the too high relevant cardiovascular disease of homocysteine.

Another aspect of the present invention is that methioninase is used to remove the homocysteine treatment.

Homocysteine is too high relevant with all kinds cardiovascular diseases.The relation of these diseases and homocysteine is that McCully found (" American Journal of Pathology " be 56:111-28 (Am.J.Pathol.) for McCully, KS, 1969) first in 1969.McCully has found the plasma homocysteine concentration of rising and the relation between the arteriosclerosis disease.The plasma homocysteine level of discovering rising of by Framingham Heart Study 1041 people being carried out recently causes the danger (" Britain's pharmaceutical journal " be Med.32:286-91 (N.Engl.J.) for Selbub, J. etc., 1995) that arteriosclerosis increases.Other research even with the medium too high and peripheral blood vessel of homocysteine blood, cerebrovascular and coronary heart disease disease connect (Kang, S. etc., " threpsology make progress annual " are 12:279-98 (Annu.Rev.Nutr.), 1992).For example angiopathy patient empty stomach homocysteine concentration ratio normal person is high by 31%.Plasma homocysteine concentration surpasses the normal value upper limit 12% and both formed dangerous 3.4 times relevant (" AMA's proceedings " be 268:877-81 (JAMA) for Stampfer, M. etc., 1992) of increasing with the trouble myocardial infarction.Discovering of the homocysteine metabolism mechanism of action; In more than 2000 experimenters; 20 example contrasts and cross-region research shows, suffers stroke or the hemopathic patient of other cardiovascular has than has the blood level of the higher high light cystine of cardiovascular disease testee.This and most of myocardial infarction patient have the true opposite of normal cholesterol level.Found the result that attracts people's attention at " internal medicine health research " (Physicians Health Study); Expection shows in the blood sample that 271 male that suffered from myocardial infarction afterwards by diagnosis took out, to have apparently higher than the average ground line level of not suffering from the suitable matched group of myocardial infarction of homocysteine on one's body before the trouble myocardial infarction.(Ueland, P. etc., " cardiovascular disease hemostasis and endothelial function " Cardiovascular Disease Hemostasis and Endothelial Function, New York:Marcel Dekler; 183-236,1992).Although have multiple condition can cause the rising of homocysteine levels,, have the relation between high-level homocysteine and the angiopathy no matter what the metabolism reason is.In a directed research, in the baboon body, bring out blood vessel injury, and with homocysteine to baboon infusion three months (Ueland, P. etc., preceding text).Measuring the interior homocysteine levels of patient's body behind the administered through oral methionine diagnoses homocysteine too high.It is 12 times of normal person (Ueland, etc., preceding text) that oral methionine excites back abnormal homocysteine PC in arteriosclerotic's body.Can measure plasma levels of homocysteine quickly and easily with HPLC mensuration.Research shows that 40% population has the homocysteine levels of rising, has the danger (Stampfer, M. and Malinow, M., " New England Journal of Medicine " (New Engl.J.Med.) 332:326-3291995) of suffering from the cardiovascular diseases.Homocysteine blood is too high to have acute effect aspect the arteriosclerosis disease bringing out, and makes individuality that the danger of suffering from myocardial infarction and cerebrovascular arranged.Emergency medical is got involved the major part that shows above-mentioned individuality has the danger of suffering from cardiovascular disease.Methioninase of the present invention can be used as selectable Therapeutic Method in the emergency medical, can reduce the people's that risk is arranged homocysteine levels at once.Two kinds of reactions of methioninase catalysis; Not only enzymolysis N-C key and γ C-S key (Hoffman in methionine but also in homocysteine; " biotechnology and biophysics " (Bioch.et Biophys.) Acta; " cancer research review " (Reviews on Cancer), 738:49-87,1984).Fig. 7 explains homocysteine and the metabolic metabolic cycles of methionine.Vitamin B-12, the circulation left side of explanation is helpful to keeping some normal homocysteine levels with methioninase treatment back among B-6 and the folate influence figure.And the level of these vitamin is not depended on the circulation right side.On the circulation right side, the existence of methionine causes excessive homocysteine levels through the Methyl transporters reaction that raises, and it can cause cancer and arteriosclerosis disease.For the abnormal patient in circulation right side, it is necessary that maintenance and short-term are used methioninase, can keep the normal level of homocysteine.For cardiovascular disease, methioninase is important to the substrate specificity of methionine and homocysteine.Methioninase can reduce the level of homocysteine precursor methionine, also can directly reduce homocysteine levels.Vitamin B-12 is used in the existing research in this area only suggestion, and B-6 and folate reduce too high homocysteine blood as therapeutic agent.As shown in Figure 7, only show that these vitamin do not correct any undesired symptom in should the circulation right side, also not reducing should the intravital homocysteine levels of the abnormal patient in circulation right side.The present invention includes with the methioninase treatment and suffer from the method for cardiovascular patient.

On the one hand, provide treatment to suffer from the method for cardiovascular patient, comprised methioninase to patient's administering therapeutic effective dose.

The treatment effective dose that is used for the methioninase of homocysteine removal is premeasuring, calculates the level that institute's phase homocysteine is removed that reaches, thereby, for example, reduce and suffer from arteriosclerotic danger.

Therefore, the dosage range of methioninase administration of the present invention is the amount that is enough to produce institute's phase effect, and institute's phase effect for example is to reduce to suffer from arteriosclerotic danger or level.Dosage should be greatly to causing side effect, like hyperviscosity, and syndrome, pulmonary edema, congestive heart failure or the like.Dosage is according to patient's age generally speaking, and symptom, sex and disease progression degree are and different, and this is that those skilled in the art can determine.

Dosage can be confirmed by the doctor under any complicated situation.

The treatment effective dose of methioninase of the present invention generally is that when with the compatible compositions administration of physiology, to be enough to make the concentration of (blood plasma) in the blood vessel or local methioninase be about 0.001 to about 100U/ml; Preferably be higher than about 0.1U/ml, more preferably be higher than the amount of 1U/ml.The general dosage of administration is decided by body weight, and is at approximately 5-1000U/kg/ days, preferably approximately 10-50U/kg/ days, and more preferably in about 20-40Ukg/ days the scope.

In a preferred method, used methioninase has basically no endotoxin, goes through like preceding text.Particularly preferably be the methioninase that uses the present invention to produce, it is from the reorganization source and be substantially free of endotoxin.

Can use the known method administration of this area ordinary technical staff, and be illustrated in this manual.

Another aspect of the present invention is to treat the patient who suffers from cardiovascular disease through the methioninase that is substantially free of endotoxin and combines with polymer of administering therapeutic effective dose.One preferred aspect, this polymer is a Polyethylene Glycol.Another preferred aspect of the present invention is that said cardiovascular disease is an arteriosclerosis.Another aspect of the present invention is narrow to the cranium arteria carotis externa, peripheral blood vessel, cerebrovascular, coronary heart disease and obliterative vascular disease patient administration methioninase.

Another aspect of the present invention has provided a kind of method through the methioninase of patient's drug treatment effective dose is treated the too high patient of homocysteine blood.Said methioninase is substantially free of endotoxin and combines with polymer such as Polyethylene Glycol.

The present invention also provides the method that reduces homocysteine levels in patient's body, comprises that wherein said methioninase is substantially free of endotoxin to the step of the methioninase of patient's drug treatment effective dose.The present invention has provided the method that cardiovascular disease takes place a kind of patient of prevention on the other hand, comprises the step to the methioninase of patient's drug treatment effective dose.One preferred aspect, have basically no endotoxin for the methioninase with the patient.Another preferred aspect, said methioninase combines with a polymer.Provide the prevention patient that the method for cardiovascular disease takes place again on the one hand, comprise that said methioninase has basically no endotoxin to the step of the methioninase of patient's drug treatment effective dose, and combine with Polyethylene Glycol.Embodiment 7 provides the example that homocysteine is removed in the body.

H. methioninase is applied to tumor imaging

The present invention provides the method for diagnosing patients in-vivo tumour on the other hand, comprises through to patient's administration methioninase, removes in patient's body ¹²The C methionine is then through giving and the patient ¹¹The C methionine enriches the intravital methionine of patient, measures at last to exist in the patient tumors cell ¹¹The C methionine, ¹¹The C methylation can be through detecting such as but not limited to positron emission fault developing (PET) method for scanning.This area ordinary technical staff is familiar with other detection cancerous cell absorption ¹¹The method of C.These methods are described in, for example, Lapela etc., " Journal of Nuclear Medicine " be 35:1618-23 (J.Nucl.Med.), and 1994; Miyazawa etc., " Journal of Nuclear Medicine " be 34:1886-91 (J.Nucl.Med.), and 1993; Leskinen-Kallio etc., " Journal of Nuclear Medicine " be 33:691-95 (J.Nucl.Med.), and 1992; Shields etc., " Journal of Nuclear Medicine " be 33:581-84 (J.Nucl.Med.), and 1992; Huovinen etc.; " Britain's cancer magazine " (Brit J.Cancer) 67:787-91,1993; Dethy etc., " Journal of Nuclear Medicine " be 35:1162-66 (J.Nucl.Med.), and 1994; Lindholm etc., " Journal of Nuclear Medicine " be 34:1711-16 (J.Nucl.Med.), and 1993; Leskinen-Kallio etc., " Journal of Nuclear Medicine " be 32:1211-18 (J.Nucl.Med.), and 1991; With Mineura etc., " Journal of Nuclear Medicine " be 32:726-28 (J.Nucl.Med.), and 1991, draw for referencial use here together.

The preferred aspect of the present invention, the methioninase that is provided has basically no endotoxin.

Another preferred aspect of the present invention is that said methioninase is and polymer, and is bonded like Polyethylene Glycol.

The accompanying drawing summary

Fig. 1 be described in detail in 6 figures do not contain methionine, do not contain homocysteine or do not contain methionine and the homocysteine culture medium in the growth of tumor cell line.

Fig. 2 specifies methioninase reduces people's lung tumor (H460) level of growth in mice xenotransplantation effectiveness.Also specified the result of 5-FU and vincristine treatment.

Fig. 3 specifies methioninase, 5-FU and vincristine to transplanting the body weight effect of human lung cancer (H460) Mus.

Fig. 4,5 and 6 shows the pharmacokinetics of taking methioninase and methionine after the methioninase to three patients.With the active percentage ratio of methioninase as the active relative percentage of the initial preparation of methioninase.With the percentage ratio of methionine as the relative percentage of taking methionine before the methioninase.

Fig. 7 specifies methioninase-homocysteine metabolism circulation.

Fig. 8 provides the nucleotide sequence and the amino acid sequence corresponding of the methioninase of the coding methioninase dna molecular of separating from pseudomonas putida.

The model of typical high expressed in Fig. 9 diagram carrier.

Figure 10 brief description obtains high-purity, does not contain the purification step sketch of endotoxic methioninase.

Figure 11 summarizes the typical purity and the response rate of RMETASE (rMETase) when using purification process as herein described.

Figure 12 A and 12B provide the example that adopts the inventive method purification rMETase product.

Figure 13 provides the activity figure of the rMETase of different dosage form.

Figure 14 A-14D provides the analytical data to the HPLC of PEG-rMETase.

Figure 15 shows the activity of the branch period of the day from 11 p.m. to 1 a.m PECT-rMETase of different PEG and rMETase.

Figure 16 provides the pharmacokinetics of PEG-rMETase in the mice body.

Figure 17 shows that rMETase is to the growth inhibited effect in the cultivation of KB3-1 cells in vitro.

Figure 18 provides the effectiveness of the KB3-1 cell of the anti-nude mice of rMETase.

Figure 19 provides the toxicity of showing rMETase with the nude mice body weight.

Figure 20 provides rMETase the toxicity of the nude mice blood cell to having transplanted the KB3-1 cell.

Figure 21 provides the toxicity of rMETase to BALB/C mice.

Figure 22 provides the toxicity of rMETase to BALB/C mice.

Figure 23 provides the toxicity assessment to methioninase among the people patient.

Figure 24 provides the pharmacokinetics evaluation to methioninase among the people patient.

Figure 25 provides and has confirmed the growth inhibiting data of rMETase to intravital H460 of nude mice and HT29.

Figure 26 provides the contrast to the sensitivity of rMETase of external normal cell and human cancer cell.

These accompanying drawings are unnecessary to be drawn in proportion, and some characteristic of the present invention can be amplified on ratio and for clear and briefly represent with the form of sketch.

Embodiment

Relate to embodiments of the invention below and be and specify and not as to special restriction of the present invention.And present known or developable these variations of the present invention later on that can predict in the scope those skilled in the art are considered in the desired hereinafter scope of the present invention.

Embodiment 1

Enlarge and produce methioninase

The pseudomonasputida strain, modified and select the bacterial strain of kalamycin resistance and high level expression methioninase as follows:

Buy ATCC 8209 pseudomonasputidas (Pseudomonas putida) and ATCC 77100 escherichia coli in October, 1993 from ATCC.ATCC 77100 is growth in the LB that contains 50 μ g/ml kanamycin (Sambrook etc., " molecular cloning, experiment guide " (Molecular Cloning:ALaboratory manual) A.1,1989).Separation quality grain pCN 51 from ATCC 77100, a kind of shuttle plasmid (Nieco etc., Gene 87:145-149 that comprises the kalamycin resistance gene that can duplicate among both pseudomonasputida (P.putida) and escherichia coli (E.coli); 1990; Here draw for referencial use), and with Triton-lysozyme method (Sambrook etc., " molecular cloning; experiment guide " (Molecular Cloning:A laboratory manual) 1.29-1.30,1989) purification.ATCC 8209 grows in the LB culture medium; And transform with pCN 51 with the standard conversion method; Said standard conversion method is as being described in for example Sambrook etc.; " molecular cloning, experiment guide " (Molecular Cloning:a laboratory manual.) 1.74-1.84, those methods in 1989.The kalamycin resistance bacterial strain is selected with kanamycin (100 μ g/ml) in the LB culture medium; And further at LB plate (Sambrook etc.; " molecular cloning; experiment guide " (Molecular Cloning:Alaboratory manual) A.1-A.4,1989) go up and in 100 μ g/ml kanamycin, to grow.Single bacterium colony is grow overnight in the LB that contains 100 μ g/ml kanamycin; Place then under the homomethionin expression of enzymes condition: 10%LB; 0.1% kaliumphosphate buffer (pH 7.2), 0.1% carbamide, 0.025% yeast extract; 0.01% magnesium sulfate and 0.25% methionine that 50 μ g/ml kanamycin are arranged were cultivated 24 hours.Measure the expression of methioninase with the standard methioninase assay method of this description explanation.Select the pseudomonasputida bacterial strain (Pseudomanas putida) and the called after AC-1 of excessive production methioninase.

Use the sweat that AC-1 pseudomonasputida bacterial strain (Pseudomonas putida) enlarges production then.The single bacterium colony of AC-1 is grown on the LB plate that contains 50 μ g/ml kanamycin.At 26 ℃ the bacterium colony of selecting was contained among the LB of 50 μ g/ml kanamycin incubation 18 hours at 5ml with 250rpm/min jolting speed.Jolt speed with 200rpm/min and the 2ml antibacterial was contained among the LB of 50 μ g/ml kanamycin amplification 6 hours at 600ml at 26 ℃.Sway at 26 ℃ 50ml antibacterial in 2 liters of LB that contain 50 μ g/ml kanamycin, be incubated overnight (18 hours) with 100rpm/min then.Let 2 liters of antibacterial (OD then ₆₀₀1.2-1.6) in 40 liters of jars, containing 10%LB, 0.1% kaliumphosphate buffer (pH 7.2), 0.1% glycerol; 0.1% carbamide, 0.025% yeast extract is in the defined medium of 0.01% magnesium sulfate and 0.25% methionine; Under the ventilated condition, 26 ℃ of growths 24 hours.OD ₆₀₀Reach 1.2-1.8 and activity and reach the 20-30 units per liter.The cell optimum density of results is 1.5-1.8OD ₆₀₀, having the 2-3mg/l methioninase, this is equivalent to 1kg wet cell/400 liter.Preferably, the well-found fermentation tank that uses about productive rate 1kg wet cell/10-20 to rise.Keep 4 ℃ of temperature, with AGT post (UFP-500-E-55 type cylinder, A/G Technology Corporation) collecting cell, then at 4 ℃, with 9krpm, with centrifugal 10 minutes of automatic refrigerated centrifuger (Sorvall Superspeed RC2-B).Collecting cell pellet then.

Then with cell suspension in extracting in the solution (20mM potassium phosphate, pH 9.0), density be 500 gram wet cells/liter, three times with cavity homogenizer (Microfluidics Corp.Model#HC 8000) smudge cells.Immediately homogenate is preserved down at-800 ℃ behind the cell breakage.The specific activity of methioninase is 0.08～0.1 unit/mg albumen in the homogenate.

The AC-1 homogenate is suspended in extraction buffer (10mM potassium phosphate, pH 7.2,10 μ M pyridoxal 5-phosphates, 0.01% beta-mercaptoethanol, 1mM EDTA and 20% ethanol), and 50 ℃ of heating 2 minutes.Heating steps can carry out the time of any length, is enough to the responsive impurity albumen of precipitation heat and keeps methioninase active.Suspension 4 ℃ with 12krpm centrifugal 30 minutes.Collect supernatant also with MilliporePrep/Scale-TFF PLHK 100k 2.5ft ²The cylinder ultrafiltration.Then pH is transferred to 7.2.

Approximately the 10L of 25-35g gross protein extracts buffer (10mM kaliumphosphate buffer pH 7.2; 10 μ m pyridoxal 5-phosphates and 0.01%J-mercaptoethanol) sample is applied to 10cm * 50cmToyopear (on the DEAE-650M post (Toso Haas Japan), this post is with 10mM kaliumphosphate buffer (pH7.2) pre-equilibration.This post washes with 10mM kaliumphosphate buffer (pH7.2) 20-30L of the 40mM potassium chloride that contains 10 μ m pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol earlier in advance, until OD ₂₈₀Be reduced to below 0.1.Then potassium chloride is increased to 300mM at the extraction buffer from initial concentration 40mM, pillar is carried out linear gradient elution.Collect 400ml and washed out fraction.Measuring the fraction that contains methioninase with the methioninase determination of activity of this description explanation also concentrates.With the 10mM kaliumphosphate buffer that contains same concentrations pyridoxal 5-phosphate and beta-mercaptoethanol, pH8.3 and 150mM potassium chloride are with the fraction pre-equilibration ratio of concentrating.

Will (buffer sample of about 1-2g gross protein that the methioninase peak of post of 5cm * 20cm) obtains be applied on DEAE-Sephadex A 50 posts from DEAE-650M; Said buffer contains 10mM potassium phosphate pH8.3; 150mM potassium chloride, 10 μ M pyridoxal 5-phosphates and 0.01%J-mercaptoethanol.With this post of 150-500mM KCl linear gradient elution in the 10mM kaliumphosphate buffer (pH8.3) that contains 10 μ M pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol, collect 150ml and wash out fraction.Concentrate the fraction of measuring with the active testing of this paper explanation that contains methioninase.Be further purified concentrated methioninase then to remove endotoxin.Through in the 10mM of 0.12M sodium chloride and pH7.1 sodium phosphate buffer, dialysing with the sample pre-equilibration.With sample be added to 5cm * 15cm Acticlean Etox resin column (Sterogen Bioseparations, Arcadia, CA) on.Then with same buffer eluting enzyme from the post, collect and have the active fraction that washes out of methioninase, measure then and wash out endotoxin content in the fraction.Table 1 provides the result of this purification process.

The analysis of the methioninase of purification on the 7.5%SDS-PAGE gel shows a 43kd protein belt, the proteinic subunit of 172kd that representative is found when analyzing the active fraction that from second post, obtains with SDS-PAGE.It is 98.7% that HPLC analyzes the purity that shows with the isolating methioninase of this method, through having only a protein peak provable.

The method for distilling of this paper explanation has produced and has had basically no endotoxic methioninase preparation of purifying basically.This area ordinary technical staff understands and can modify the method for distilling of explanation that these changes comprise within the scope of the present invention.

Embodiment 2

People's tumor is to the dependency of the methionine level of rising

The methionine dependency often occurs under the human cancer situation, is used to test the methionine dependency from 20 kinds of NCI human tumor cell lines of all major organs systems.All 20 cell lines all find it is that methionine is dependent.Tested fresh people's tumor sample in addition, also found it is that methionine is dependent.Normal cell system is not that methionine is dependent.

Fig. 1 has provided the result of a this test.In Fig. 1, normal cell strain and tumor cell line are grown in Eagle ' s MEM with the serum of 10% dialysis, and the serum of said dialysis is one of following sample: do not contain homocysteine and contain methionine (MET ⁺HCY ^-) sample; Do not contain methionine and contain homocysteine (MET ^-HCY ^-) sample or do not contain methionine and do not contain homocysteine (MET ^-HCY ^-) sample.Containing methionine (MET) culture medium; No MET culture medium; Contain in homocysteine (HCY) culture medium and, contain in homocysteine (HCY) culture medium and, screen kidney in the no HCY culture medium at no MET in no MET culture medium; Colon, lung and prostate cancer cell line and melanoma and normal fibroblast strain.Cell is hyclone after being supplemented with 10% dialysis, growth in the Eagle ' s minimum essential medium (MEM culture medium) of 10 μ m hydroxocobalamines and 100 μ m folic acid.This culture medium adds or do not add methionine: MET+HCY with following mode: the L-methionine that does not contain homocysteine with 100 μ m is added this culture medium.METHCY+: do not contain the D of methionine with 200 μ m, the L-homocysteine is added this culture medium.METHCY: this culture medium does not contain methionine and does not contain homocysteine.Hyclone is to phosphate buffered saline (PBS) (PBS) (serum: PBS is 1: 12) dialysis 10 times.

Cell proliferation is measured in the reductive absorption of metabolism through measure dyestuff XTT at 450nm.Through inoculating 5 * 10 in every hole ³With 2 * 10 ⁴Cell within the individual cell scope is grown on 48 hole plates by cell.(2, two (2-methoxyl group-4-nitro-5-sulfophenyl)-5-[(phenyl amino) carbonyl] of 3--2H-tetrazolium hydroxide is used to measure cell proliferation to be reduced into the XTT of water solublity first

product by metabolism in the living cells.Measure the absorption value of reduzate at the 450nm place.Temperature prepares 1mg/ml XTT in advance in 37 ℃ of PBS, preparation 5mM azophenlyene N-metilsulfate (PMS) in PBS.Fresh XTT solution and PMS storage solution are with the mixed of every 1ml XTT 511PMS.Every hole 1ml culture medium of packing into.In every hole, add the blended XTT of 0.25ml.Inoculate after 3 hours the absorption value of reading reductive XTT with Hitachi U-2000 spectrophotometer at the 450nm place.The initial OD that measured an XTT after 24 hours in per three days that cultivates of cell.At MET ⁺HCY ^-Or MET ^-HCY ⁺In, foreskin fibroblast strain FS-3 and HS-68 equally breed basically.But present disclosure has been reported a new discovery, and promptly in MET HCY culture medium, two kinds of fibroblast strains are bred after a certain degree and kept at least 16 days.

Table 2 has specified the mensuration result of the cell proliferation of 20 kinds of tumor cell lines.In this is measured, with 5 * 10 ³-2 * 10 ⁴The cell suspending liquid of individual cells/well was cultivated 24 hours in the MEM that is containing 10% hyclone on the 48 hole flat boards.Be divided into three groups with Hanks alkaline salt solution (HBSS) with behind twice of the cell washing.First group: cell is at MET ⁺HCY ^-Cultivate in the culture medium; Second group: cell is at MET ^-HCY ⁺Cultivate in the culture medium; The 3rd group: cell is at MET ^-HCY ^-Cultivate in the culture medium.Cell is with 95% air/5%CO ₂Cultivate in the incubator of ventilation.Changed a subculture in every 2-3 days.In table 2, what cell line growth was vigorous representes with a+++, and to some extent representing with a++ of growth, slightly representing with a+ of some growth, growth does not represent with a-.Only about half of tumor cell ties up to no MET and contains in the culture medium of HCY and can not grow, and other cell line is grown in various degree, and with identical culture medium and in containing the culture medium of MET, normal cell can be grown.Although do not have in the culture medium of HCY at no MET; The normal cell strain can grow into to a certain degree, and the back keeps 2 weeks or longer time; But all tried 20 kinds of tumor cells tie up in this culture medium dead, this show might be all cancerous cell killed selectively through removing MET.This embodiment provides evidence proof especially to use methioninase as therapeutic agent for treatment of cancer, and the methionine dependency possibly be general and target optionally.

Embodiment 3

Methioninase is to the inhibitory action of people's tumor growth in the mice heteroplastic transplantation model

For testing methioninase, used the mice heteroplastic transplantation model to slowing down the drug effect of tumor growth.People's pulmonary carcinoma tumor (H460) is transplanted in the subcutaneous tissue of nude mice.One group of per four mice are divided into four groups with mice.Transplant after four days, through per 4 hours to intraperitoneal injection give A group 0.4ml buffer (0.12M sodium chloride, the 10mM sodium phosphate, pH7.1); Gave B group methioninase (1 unit/g) through per 4 hours to intraperitoneal injection; Gave C group 5-FU (60mg/kg) through per 4 days to intraperitoneal injection; Give D group vincristine (VCR) (0.9mg/kg) through intraperitoneal injection in per 4 days.Tumor size of measurement in per two days and body weight.The result is shown in table 2 and 3.The result shows, with methioninase treatment having slowed down greatly growth of H460 people's nonsmall-cell lung cancer in the nude mouse.In these experiments, mice feeds the normal diet of advancing not remove methionine.The drug effect of the anti-H460 of methioninase is opposite with the vincristine treatment with 5-fluorouracil fully, and back two kinds of medicines do not have the activity of anti-this tumor.Give not cause in the treatments in 10 days of methioninase 40-120 active unit/sky and lose weight that this shows that it has hypotoxicity.On the contrary, vincristine causes loses weight, and shows toxic.Therefore, methioninase is a kind of little high efficiency anti-tumor agent of toxicity that new tumor-selective action method is arranged, and proves the clinical effectiveness of anti-people's solid tumor potentiality.

Embodiment 4

Preparation PEG-methioninase

Molecular weight is that 5000 methoxy Polyethylene Glycol succinimdyl carbonate (M-SC 5000PEG) is dissolved in the 20mM sodium phosphate buffer (pH8.3), and concentration is between 2mM and 20mM.The mol ratio of M-SC5000PEG and methioninase changed from 6: 1 to 240: 1.PEGization is reflected at reaction buffer, and (the 25mM sodium phosphate buffer carried out 30 minutes to 60 minutes at 4 ℃ or 20 ℃ in pH8.3).(the 0.14M sodium phosphate buffer is pH6.5) 0 ℃ of cessation reaction with stop buffer.Remove unreacted M-SC 5000PEG according to the gel permeation chromatography of this paper explanation then.Use 30k AmiconCentriprep concentrator, in 0.12M sodium chloride and 10mM sodium phosphate (pH7.2), be mixed with final PEG-methioninase.Through the filtration of 0.2 μ m microgranule film filter the PEG-methioninase is sterilized then.This PEG-methioninase can be saved to 6 months and loss of activity not at-70 ℃.

The mensuration methioninase is active.PEG-methioninase reservation at least 60% is the activity of PEGization methioninase not.According to the explanation of Laemmli and Sambrook (Laemmli, " maturation " be 227-680 (Mature), 1970; Sambrook etc.; " molecular cloning; experiment guide " (Molecular Cloning:a laboratory manual) 18,47-18.59,1989); Analyze the PEG-methioninase through natural with sds polyacrylamide gel electrophoresis, and carried out following improvement: carry out PEG-methioninase electrophoresis in 7.5% polyacrylamide precast gel in being with or without the 0.2MTris-glycine buffer (pH8.3) of SDS.Gel is used Coomassie blue stain, and discolors with 40% methanol, 10% acetic acid.Although visible very little electrophoresis band when the PEG-methioninase is administered on the natural gel, can not see the electrophoresis band of unmodified methioninase in sds gel.

Analyze the PEG-methioninase with the HPLC solvent resistant column as follows then: with PEG-methioninase (20 μ l; 1-2mg/ml is added to 0.2M phosphoric acid with the sample injector of one 20 μ l and receives on Progel-TSK G3000SW (Supelco) post in the buffer (pH7.2), and with 0.2M sodium phosphate buffer (pH7.2) with flow velocity 1.0ml/min eluting.Only detect a protein peak, promptly do not contain the PEG-methioninase peak of unreacted PEG; Detection is less than the methioninase peak of PEGization not.The purity of PEG-methioninase approximately is 100%.

A) time course of PEGization reaction research

The following time course of measuring Polyethylene Glycol and methioninase combination: 0.07mM methioninase and 0.4mM-SC 5000PEG reacted 2 hours at 4 ℃ of reaction different times in 0.2M sodium phosphate buffer (pH8.3) at the most.At each time point with 0.2M sodium phosphate (pH6.5) cessation reaction.Remove unreacted PEG with the centrifugal preparation concentrator of 30kAmicon.The result sees table 3.

The PEG-methioninase is analyzed in explanation according to this paper on natural and SDS-PAGE gel: measured HMW and taken out of existing; And in table 3, indicate-,+-,+or ++ symbol;-show do not have PEG-methioninase or methioninase; There are a small amount of PEG-methionine or methioninase+-show, ++ showing has a large amount of PEG-methioninases or methioninase.Table 3 also shows the activity of PEG-methioninase or methioninase.

^*The M=methioninase ^*P=M-SC 5000-PEG.

B) with the concentration dependent of M-SC 5000PEG to methioninase PEGization

0.07mM the M-SC 5000PEG of methioninase and various concentration (0.4mM to 4.0mM) reacts in 0.2M sodium phosphate buffer (pH8.3).Mol ratio is 1: 6～1: 60.Be reflected under the identical condition of embodiment a and carried out 60 minutes at 4 ℃.

The result sees table 4.

^*M: methioninase ^*P:PEG. ^{* *}The P-M:PEG-methioninase.

C) pharmacokinetics of PEG-methioninase

The M-SC 5000PEG that methioninase PEG turns usefulness into is respectively 1: 6 (P1) and 1: 12 (P2) to the mol ratio of methioninase.Be reflected under the condition identical in the preceding text (b) and carry out.The methioninase of the P1 of purification and P2 and unmodified injects the tail vein of three groups of different mices respectively.0,1,2,3,4 and 20 hours blood samplings carry out determination of activity to methioninase and PEG-methioninase, and measure the level of removing methionine, and the result sees table 5.

M: methioninase. ^*P1, the P2:PEG-methioninase

Embodiment 5

Use the PEG-methioninase in the Cavia porcellus body, to carry out the antigenicity experiment:

Methioninase and M-SC 5000PEG be respectively with mol ratio 1: 60, and 1: 120 and 1: 240 were 20 ℃ of reactions 30 minutes.Other condition is identical with preceding text (a).Through the methioninase active testing, electrophoresis and HPLC analyze the PEG-methioninase.Detection is less than the band or the peak of unmodified methioninase.Therefore, the PEG-methioninase is unique enzyme source.

Cavia porcellus intraperitoneal administration 2mg methioninase or PEG-methioninase were administered three times altogether in per 2 days.Behind the fortnight, intravenous administration 4mg methioninase or PEG-methioninase.In this experiment respectively with 0.2 sodium phosphate buffer and BSA as feminine gender and positive control.Criterion evaluation result according to table 6.The result sees table 7.According to the Cavia porcellus in vivo test, the PEG of methioninase turns into the composition that has produced utmost point low antigenicity.It is more responsive that known Cavia porcellus and people compare antigen.

Table 6

The evaluation criteria of PEG-methioninase antigenicity research in the Cavia porcellus body

Evaluation criteria

(-) feminine gender: no change

(+) is light to be become: 1-4

(++) moderate: 1-10

(+++) serious: 1-19

(++ ++) death: 20

The result shows that PEG-methioninase and bovine serum albumin (B.S.A) are opposite, and methioninase is no antigen in the Cavia porcellus body, and this shows that the PEG-methioninase is nonantigenic when to the mammal administration.

Embodiment 6

The administration methioninase is used for the human cancer treatment

Begin to carry out methioninase I stage dose escalation study and measured methioninase toxicity, pharmacokinetics and maximal allowance dose.Through give 2 advanced breast cancer patient intravenouss (iv) infusion gave 2 hours infusions of 5000 units (0.5g) and 10000 units (1.0g) methioninase in 2 hours.From 0 to 24 hour with frequent interval blood sampling and urine sample.Carry out the toxicity assessment according to the WHO hierarchical system.The level of methioninase and methionine from serum and obtain pharmacokinetic data.

Do not find acute clinical toxicity, all toxicity criterions of mensuration have 0 minimum level.Patient 1 is respectively 2 hours and 3.2 hours with the half-life of the interior methioninase of patient's 2 bodies.Begin to remove the serum methionine behind the infusion in 30 minutes, and after infusion is accomplished, kept 4 hours.Minimum serum methionine level is respectively to handle 35% and 19% of preceding level in patient 1 and patient's 2 bodies.The result is shown in table 8-10 and Fig. 4-6.As follows the patient is treated:

The patient 1: the date: on December 14th, 1994.Diagnosis: advanced breast cancer, axillary gland and lung shift.The women, 46 years old.Infusion 200ml administration 5000 units (0.4g) are according to the methioninase of method for distilling (embodiment 1) purification in 2 hours angular veins.Per 2 hours of before the treatment and treatment back is until back 20 hours blood samplings of treatment.Measure methionine and methioninase level and calculate level relatively, Fig. 4.

The patient 2: the date: February 2 nineteen ninety-five.Diagnosis: advanced breast cancer, axillary lymphatic metastasis.The women, 54 years old.Infusion 400ml administration 10000 units (0.8g) are according to the methioninase of method for distilling (embodiment 1) purification in 2 hours angular veins.Per 2 hours of before the treatment and treatment back is until back 20 hours blood samplings of treatment.Measure the level of methionine and methioninase and calculate level relatively, Fig. 5.

The patient 3: the date: October 15 nineteen ninety-five.Diagnosis: advanced breast cancer, axillary lymphatic metastasis.The women, 45 years old.Infusion 1000ml administration 20000 units (1.6g) are according to the methioninase of method for distilling (embodiment 1) purification in 10 hours angular veins.The per 2 hours blood samplings in before the treatment and treatment back until back 20 hours of treatment, are measured methionine and methioninase level, and are calculated level relatively, Fig. 6.

The 50% high methioninase serum levels that keeps maximum horizontal in patient 36 hours behind infusion.Through 10 hours infusion, methionine was removed and is surpassed 200 times, reduces to 0.1 μ M from 23.1 μ M., all toxicity criterions do not find clinical toxicity in measuring.

Carry out the toxicity assessment aspect four according to the WHO toxicity criterion: a. medical history and diagnostic data; B. physical examination; C. Laboratory Evaluation; With d. pharmacokinetics assessment (methioninase and methionine content in the serum).The result proves that methioninase of the present invention is avirulent and can be used for reducing the methionine level shown in table 8-11 and Fig. 4-6 in human body.

Embodiment 7

The removal of homocysteine in the body

Render a service through as follows this enzyme of mice administration having been proved that methioninase of the present invention is used to remove in the body of homocysteine: the 4 unit methioninases of giving the method purification of injection embodiment 1 in the mouse peritoneal.After injecting about 1 hour, carry out anti-phase FPLC through Mus serum and analyze and measure methionine, the blood sample concentration of homocysteine and cysteine 50 μ l PITC derivatizations.With chromatogram and the deutero-raw sample methionine of PITC, the chromatogram of homocysteine and cysteine standard sample is compared.The susceptiveness of test is about 0.5 μ M methionine.

As shown in table 11, to compare with cysteine with the high hemiamic acid of untreated mice contrast, methioninase has homocysteine removal activity in the significant body, and has no the removal effect of cysteine in the tangible body.

Embodiment 8

Methioninase is used for tumor imaging

Illustrated like this description, through the methioninase of administration purification, remove in patient's body [ ¹²C] methionine.Preferably, this methioninase does not have endotoxin.Through 4～8 hours administration methioninases (10000-20000 unit) of intravenous input.Give then about 5-50mCi [ ¹¹C] methioninase.Use then positron emission tomography (PET) imaging measure patient's cell [ ¹¹C] absorption of tracer.Known method is worth quantitative assay tracer intake through normalized absorption value (S, U, V) and the dynamic constant (Ki) that flows into by one of ordinary skill in the art.The interior elevated levels of cell [ ¹¹C] methionine shows that these cells possibly be tumor cells.Tumor cell preferentially take in [ ¹¹C] methionine provides a kind of method that in normal cell, optionally detects tumor cell.

Specifying of preceding text comprises that instantiation and embodiment are not as limiting in order to specify the present invention.Be no more than true spirit of the present invention and scope, can carry out multiple other variation and modification.

Embodiment 9

The nucleic acid molecules that separates the coding methioninase

The PCR reaction of methioninase gene clone insert:

With the genomic DNA of the deutero-pseudomonasputida AC-1 of ATCC8209 as template; The primer is following:

T1:5 '-GCCGGTCTGTGGAAT AAGCT-3 ' (justice is arranged),

Hind?III

T2:5 '-CCAG GGTCGACTCCAGCGCC-3 ' (antisense)

Sal?I

The PCR reaction condition is following: 95 ℃ of degeneration for the first time 10 minutes, then 94 ℃ of degeneration 30 seconds, 60 ℃ of annealing 30 seconds; Extended 2 minutes at 72 ℃, carry out 5 circulations, annealed 30 seconds for 60 ℃ then 94 ℃ of degeneration 30 seconds; Extended 1.5 minutes at 72 ℃ then, carry out 25 circulations; Then 75 ℃ of last stretching, extensions 10 minutes.Pcr amplification product has two electrophoresis bands, collects the wherein band of 1365bp, and the purification thing is insert ONCase-1DNA.

Clone and conversion

ONCase-1DNA is connected at EcoR VT-cloning site with pT7Blue T-carrier (Novagen).With standard method pONCase-1DNA is transformed in the DH5-α bacterial cell.

Determined dna sequence

Carry out determined dna sequence with T7DNA polymerase and dideoxy nucleotide cessation reaction.Use primer migration method.With [ ³⁵S] the dATP labelling.On 6% polyacrylamide wedge shape that contains 8M carbamide or non-wedge-shaped gel, analyze sequencing reaction.Load the DNA sample with the ACGT order.Through Mac carrier analyzing DNA sequence.Fig. 8 provides DNA sequence and corresponding amino acid sequence.

Embodiment 10

The high-expression clone of RMETASE

The PCR reaction of the insert of methioninase expression cloning:

As template, the primer is following with the pONCase-1 clone:

T14.5 '-GGAATTC CATATGCACGGCTCCAACAAGC-3 ' (justice is arranged)

NdeI

T15.5 '-AGTCAT CCTAGGTCACATCATCATCATCATCATGGCACTCGCCTTGAGTGC-3 ' (antisense) BamHI

T18.5 ' AGTC-AT CCTAGGTCAGGCACTCGCCTTGAGTGC-3 ' (antisense)

BamHI

The PCR reaction condition is following: 95 ℃ of degeneration for the first time 10 minutes, then 94 ℃ of degeneration 1 minute, 56 ℃ of annealing 1.5 minutes, and 72 ℃ of extensions 2 minutes, carry out 5 circulations; 94 ℃ of degeneration 30 seconds,, extended 1.5 minutes at 72 ℃ then then, carry out 20 circulations 56 ℃ of annealing 30 seconds; Then 72 ℃ of last extensions 10 minutes.Collect and two pcr amplification products of purification ONCase-2 (1238bp), ONCase-3 (1220bp) band.

Clone and conversion

Digest ONCase-2 and ONCase-3DNA with Nde I and Bam HI, and be connected with Bam HI cloning site at Nde I with the pT7.7 carrier.With standard method PONCase-2 and PONCase-3DNA sequence are transformed in BL21 (DE3) bacterial cell then.

Select pAC-1 and pAC-2 clone

Select positive colony containing on the flat board of ampicillin.4 ℃ preserve 24 hours after, the positive colony of expressing high-level RMETASE has and significantly is used to the pink differentiating and select.Measure the expression of the methioninase of positive colony through activity analysis.Two kinds of high-expression clones selecting are the pAC-2 clones that comprise the pAC-1 clone of ONCase-3 and comprise ONCase-2.

Make up pAC-3 clone and pAC-4 clone

Obtain tetracycline resistance gene from pBR322 at AvaI and Cla I site.Ava I end is added to flush end, and is connected with pAC-1, and said pAC-1 is by BamHI and ClaI digestion with restriction enzyme, and the BamHI end is mended is flush end.Select the positive colony that becomes pink colour 4 ℃ of reservations after 24 hours from the flat board that contains tetracycline.Measure high expressed RMETASE clone through activity analysis, and called after pAC-3 clone.

Ava I and Hind III have also obtained tetracycline resistance gene in the site from pBR 322.Ava-1 end charges into a flush end, and be connected with the pAC-1 of Cla I digestion with restriction enzyme with Hind III, Cla I holds and charges into a flush end.Select the positive colony that becomes pink colour 4 ℃ of preservations after 24 hours from the flat board that contains tetracycline.Measure high expressed RMETASE clone with activity analysis, and called after pAC-4 clone.Table 12 and Fig. 9 provide various high level expression clones.

Embodiment 11

The Fermentation of RMETASE expression cloning

In the Terrific Broth culture medium that contains ampicillin (100 μ g/ml) or tetracycline (10 μ g/ml), at 28 ℃ or 37 ℃, 400rpm sways, and in 6L culture bottle or fermentation tank, cultivates the expression cloning of RMETASE.

Embodiment 12

The purification of RMETASE

Figure 10 and 11 provides the sketch map of purification process.

(1) processing before the sample upper prop

Collected antibacterial at 4 ℃ in centrifugal 10 minutes with 800 * g; Then the antibacterial pellet is suspended in and extracts solution (20mM potassium phosphate (pH9.0); 10 μ m pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol), and with cavity type homogenizer (Microfluidics Corp.model#HC 8000) fragmentation.At 50 ℃ homogenate was heat-treated 1 minute then.With automatic refrigerated centrifuger (SORVALL Superspeed RC2-B) 4 ℃ with 13rpm with centrifugal 30 minutes of suspension.Collect supernatant then.Then through Milliporeprep/Scale-TFF PLHK 100k 2.5ft ²Cylinder is with buffer (10mM potassium phosphate, pH8.3) ultrafiltration.Through ultrafiltration pH is transferred to 7.2.

(2) column chromatography condition

First post: DEAE agarose FF

Post: XK 100/60, highly: 32cm, volume: 2.5L

Solution: [A] contains the 40mM potassium chloride of 10 μ m pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol, 10mM potassium phosphate (pH7.2).

[B] contains the 200mM potassium chloride of 10 μ m pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol, 10mM potassium phosphate (pH7.2).

Flow velocity: 5ml/min

Sample: approximately 100-200g gross protein (10-20mg/ml) is applied on first pillar.

Gradient: [1] is washed to OD with the solution A of about 10 volumes in advance ₂₈₀Drop to and be lower than 0.1.

[2] gradient: 20%-100% solution B

Fraction: collect the 200ml elutriated fraction.Differentiate that through activity analysis the fraction that contains rMETase is also concentrated.

Second post: DEAE agarose FF

Post: XK 50/30, highly: 25cm, volume: 500mL

Solution: [A] contains the 100mM potassium chloride of 10 μ m pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol, 10mM potassium phosphate (pH8.3).

[B] contains the 200mM potassium chloride of 10 μ m pyridoxal 5-phosphates and 0.01% beta-mercaptoethanol, 10mM potassium phosphate (pH8.3).

Flow velocity: 5ml/min

Sample: at the 100mM potassium chloride that contains 10 μ m pyridoxal 5-phosphates, the protein (2-4mg/ml) that about 10-20g is total is applied on second pillar after dialysing 24 hours in the 10mM potassium phosphate (pH8.3).

Gradient: [1] is washed to OD with the solution A of about 5 volumes in advance ₂₈₀Drop to and be lower than 0.05.

[2] gradient: 0%～60% solution B.

Fraction: collect the 200ml elutriated fraction.Differentiate that through activity analysis the fraction that contains rMETase is also concentrated.

The 3rd post: Sephacryl S-200HR

Post: Hi Prep 26/60, volume: 320ml.

Solution: 10mM sodium phosphate (pH7.2) solution of 0.15M sodium chloride.

Flow velocity: 1.2ml/min

Sample: about 10ml concentrating sample.(at 0.15M sodium chloride, dialysis is 12 hours in the 10mM sodium phosphate (pH7.2)) are applied on the 3rd post.

Fraction: differentiate elutriated fraction and the collection that contains rMETase through yellow color and activity analysis.

The 4th post:

The rMETase of the purification of 100-200ml volume (10-20mg protein/ml) be added on 500ml

post; (the 10mM sodium radio-phosphate,P-32 solution of 0.15M sodium chloride, pH7.2) eluting is to remove endotoxin with elution buffer.

renewable use; And available 1M sodium hydroxide is clean, and can autoclaving.

Concentrate whole eluent

Concentrate final eluent with the centrifugal preparation thickener of 30K Amicon.The preparation of the rMETase of purification is a 0.15M sodium chloride, 10mM sodium phosphate, pH7.2.

purification rMETase.Histidine: on Ni++ agarose gel post, carry out chromatography

Before the upper prop after the pretreatment, the cell homogenates thing is suspended in the binding buffer liquid (the 5mM imidazoles, 0.5MNaCl, 20mM Tris-HCl, pH7.9).Then with 10 volume binding buffer liquid flushing pillar, then with 6 volume lavation buffer solutions (60mM imidazoles, 0.5M sodium chloride, 20mM Tris, HCl, pH7.9) flushing.Eluting through taking place in 6 volume elution buffers (1M imidazoles, 0.5M NaCl, 20mM TrisHCl pH7.9) behind the pillar.Differentiate the fraction that contains rMETase through yellow color, and collect.

Embodiment 13

Measure the purity of rMETase with HPLC

Post: SUPELCO, 8-08541, Progel TM-TSK, G 3000-SWXL, 30cm * 7.8mm.

Elute soln: the 10mM sodium phosphate buffer (pH7.2) of 0.15M sodium chloride.

Flow velocity: 1ml/min

Sample: 20 μ l (0.1-1mg/ml)

The embodiment that produces rMETase sees Figure 10 and 11.Purification is seen Figure 12.

Embodiment 14

The preparation that contains RMETASE (rMETase) of crystallization and lyophilized form

Pharmaceutical solutions:

With the concentration of 10-20mg/ml, preparation rMETase solution, said solution is 0.15M sodium chloride, 10mM sodium phosphate buffer (pH7.2).The stability of rMETase is seen Figure 13.

Crystal form:

With the rMETase (10-20mg/ml) in 0.15M sodium chloride and the 10mM sodium phosphate buffer (pH7.2) with Sephadex G-25 (the DNA level, ultra-fine, Sigma) post desalination.This solution is freezing in dry ice and acetone bath, uses Verdis Freeze Mobile 24 then, in 100 millibars of vacuum-80 ℃ of crystallizations 72 hours.

Lyophilized form:

RMETase (10-20mg/ml) in 0.15M sodium chloride and 10mM sodium phosphate buffer (pH7.2) is freezing in dry ice and acetone bath, and with Verdis Freeze Mobil 24 in 100 millibars of vacuum-80 ℃ of lyophilizing 72 hours.

Active testing:

Test is to contain among the 50mM phosphate buffer pH8.0 of 10 μ M pyridoxal 5-phosphates and 10mM methionine at 37 ℃ at the 1ml volume, carries out 10 minutes with not commensurability enzyme.Add 0.5ml 4.5%TCA cessation reaction.Suspension centrifugal 2 minutes with 15K rpm.With the 0.5ml supernatant with 0.5ml 0.05%3-methyl-2-[4-morpholinodithio quinoline ketone hydrazone and 1ml 1M sodium acetate (pH5.2) solution 50 ℃ of incubations 30 minutes.Use spectrophotometer at OD then ₃₃₅Measure α-butanone acid group.Method with Lowry Reagent test kit (Sigma) is measured proteinic amount, calculates specific activity with unit/mg protein.

Relatively rMETase is active, and the result shows does not have big difference between the different preparations.

Embodiment 15

The chemical modification of RMETASE (PEG-rMETase)

In the 10mM of 0.15M sodium chloride sodium phosphate buffer (pH7.2) with the rMETaes of the concentration between 0.1M and 0.2M preparation purification.Activity approximately is 20 units/mg.

The M-SC 5000PEG (Methoxy-SC-PEG, MW 5000, available from Shear water Polymer Company) of molecular weight 5000 is dissolved in the 20mM sodium phosphate buffer (pH8.3) with the concentration between 2mM and the 20mM.The mol ratio of M-SC 5000PEG and rMETase changed from 10: 1 to 120: 1.

PEGization be reflected at reaction buffer (the 25mM sodium phosphate buffer, pH8.3) in, carried out 60 minutes at 20 ℃.(the 0.14M sodium phosphate buffer is pH6.5) 0 ℃ of cessation reaction with stop buffer.Remove unreacted M-SC 5000PEG with 30K Amicon Centriprep concentrator then.With 30KAmicon Centriprep concentrator when centrifugal in 0.15 sodium chloride and 10mM sodium phosphate (pH7.2) the last PEG-methioninase of preparation.

The testing in vitro of PEG-rMETase:

Use activity analysis, electrophoresis and HPLC analyze PEG-rMETase, Figure 14-16.

Activity analysis:

The activity of PEG-rMETase be unmodified rMETase active 80% to 20% between.

Electrophoresis:

In invariance and SDS-PAGE, PEG-rMETase is carried out electrophoresis.

HPLC analyzes:

PEG-rMETase is added on the solvent resistant column, does not measure the peak of original rMETase, only find the peak of PEG-METASE.Retention time (RT) shortens with the increase of PEG and rMETase molecular proportion.

The pharmacokinetics of PEG-rMETase:

The no endotoxic PEG-rMETase of purification is injected the tail vein of mice.Per 2 hours blood sample collections.Measure the level (Figure 16) of rMETase through activity analysis.

Embodiment 16

The drug effect of RMETASE and toxicity

The growth in vitro of rMETase pair cell suppresses

In the RPMI that adds 10%FBS 1640 culture medium, cultivate KB3-1 cell (people's squamous cell cancer).The rMETase that in culture medium, adds various concentration.And at 37 ℃, 5%CO ₂Incubation under the condition.At OD ₅₇₀Relative cell number is measured at the place.The result proves the effective cell growth inhibiting of rMETase (Figure 17).

RMETase suppresses the growth in vitro of H460 and HT29

(growth is 4 days in the 0-4 unit/ml), counts survivaling cell then at variable concentrations rMETase for human lung carcinoma cell H460 and human colon cancer cell HT29.Real examination triplicate.The result proves that rMETase effectively suppresses the cell growth (Figure 25) of H460 and HT29.

External relatively normal cell and human cancer cell are to the sensitivity of rMETase

, compare as tester with normal HFF HS-68 and human keratinocyte with human colon carcinoma SW-620 and human lung carcinoma cell H322m.Cell is with variable concentrations (counting survivaling cell number after 0-4 unit/ml) rMETase grew three days.The experiment triplicate.The result proves that SW-620 and H322m cell are dead when being 1 unit/ml in rMETase concentration, and normal person HS-68 cell is still survived, and just the speed of growth slows down.Normal HFF and lung carcinoma cell compare rMETase also has the sensitivity (Figure 26) much little.

The growth inhibited of rMETase pair cell in the nude mouse

Give Balb/c nu/nu, female, eight one group injected in mice 2 * 10 ⁵Individual cell.Contrast: normal saline.I group: 30 rMETase of unit, II group: 100 rMETase of unit; From the 5th day to the 14th day, twice intraperitoneal injection in a day.Measure tumor size and body weight.The 18th day blood sampling.The result proves that rMETase suppresses tumor growth effectively and do not lose weight, and to hemopoietic have no effect (Figure 18-22).

RMETase is to the growth inhibited of H460 and HT29 cell in the nude mouse

Subcutaneous (S.C.) transplants respectively, and 10 ⁶Individual H460 cell and HT29 cell, from the 2nd day to the 16th day through intraperitoneal injection twice administration every day, 40 units or 100 rMETase of unit.Matched group twice intraperitoneal injection normal saline every day 0.2ml.Killed mice on the 16th day.The result proves that rMETase effectively suppresses tumor growth and do not lose weight, and does not influence hemopoietic.

The phase I clinical trial of the pilot scale of purified recombinant METASE

The patient 1, women, 50 years old, IV primary breast cancer and lymphatic metastasis, the rMETase of 10 hours administration 10000 units (0.5g) of intravenous infusion.

Patient 2,48 years old, woman, IV primary breast cancer and lymphatic metastasis, the rMETase of 24 hours administration 5000 units (0.25g) of intravenous infusion.

Patient 3,56 years old, woman, III phase renal carcinoma, 24 hours rMETase of administration 10000 units (0.5g) of intravenous input.Table 13.Recording body inspection, before the treatment, blood sampling every two hours during the treatment is until infuse the 48th hour (as indicated) afterwards.Measure according to the WHO standard chamber of experimentizing.

The result shows that the rMETase level raises all patients immediately in transfusion beginning back, and during infusing, keeps high level.Ground line falls back in patient methioninase level after 48 hours.The result shows that transfusion beginning back rMETase level raises immediately, in the time of 10 hours, peaks.Transfusion stops back 8 hours, and the methioninase level is 50% of a peak value, and still keeps 20% of peak value in back 16 hours in transfusion.According to WHO standard assessment lab testing result, show that patient 1,2 or 3 does not have acute toxic reaction.Table 14 with 15 with Figure 23 and 24.The result shows that rMETase does not cause any toxicity.

Claims (36)

1. the methioninase of a chemical modification, it contains the methioninase that is attached on the polymer.

2. the methioninase of the chemical modification of claim 1, polymer wherein is a polyalkylene oxides.

3. the methioninase of the chemical modification of claim 2, polyalkylene oxides wherein is a Polyethylene Glycol.

4. the methioninase of the chemical modification of claim 1, methioninase wherein is substantially free of endotoxin.

5. isolated crystal type methioninase.

6. isolated freeze-dried type methioninase.

7. isolated methioninase has the specific activity of about at least 20 unit/mg and the purity of being measured with the HPLC analysis about at least 95%.

8. therapeutic combination, it contains among the claim 1-7 that treats effective dose each methioninase.

9. method of treating tumor patient, it comprises the step that the methioninase compositions of the claim 8 of treatment effective dose is taken to the patient.

10. the method for a diagnosing patients tumor, it comprises the following steps:

A) to remove the patient intravital through take methioninase to the patient ¹²The C methionine;

B) through taking to the patient ¹¹The C methionine comes to ample supply methionine in patient's body; With

C) exist in the mensuration patient interior tumor cell ¹¹The level that the C methionine raises.

11. the method for claim 10, methioninase wherein are the compositionss of claim 8.

12. a method of giving patient treatment or angiocardiopathy preventing, it comprises the step of taking the methioninase of treatment effective dose to this patient, and methioninase wherein is substantially free of endotoxin.

13. the method for claim 12, methioninase wherein are the compositionss of claim 8.

14. the method for claim 12, cardiovascular disease wherein is an arteriosclerosis.

15. a method that reduces patient's homocysteine content, it comprises the step of taking the methioninase of treatment effective dose to this patient, and methioninase wherein is substantially free of endotoxin.

16. the method for claim 15, methioninase wherein are the compositionss of claim 8.

17. the methioninase of encoding efficiently express model; This model contains the nucleotide sequence that operability connects the coding methioninase of promoter sequence; Wherein this promoter sequence can guide the expression of methioninase in the host cell, makes that the expression of methioninase is about the 5-75% of the total protein produced of this host cell.

18. the expression model of claim 17, wherein this promoter is a kind of promoter of RNA polymerase.

19. the expression model of claim 18, wherein this promoter is the promoter of t7 rna polymerase.

20. the expression model of claim 17; Wherein the nucleotide sequence of this coding methioninase is from being selected from pseudomonasputida (Pseudomonas putida); Trichomonal vaginitis (Trichomonas vaginalis), isolating in the organism in nippostrongylus brasiliensis (Nippostrongylus brasiliensis) and the fusobacterium (Fusobacteriumsp.).

21. the expression model of claim 20, nucleotide sequence wherein be provide among Fig. 8 with its degeneracy form.

22. the expression model of claim 21, wherein the nucleotide sequence of the degeneracy form of Fig. 8 comprises the sequence that is provided among reformed Fig. 8, and this sequence is included in one or more codons commonly used in the escherichia coli (E.coli.).

23. host cell with the expression model conversation of claim 17.

24. the host of claim 23, host wherein is escherichia coli.

25. the host of claim 24, host wherein are escherichia coli (E.coli.) BL21 (DE3).

26. a carrier, it contains the expression model of claim 17.

27. an isolated nucleic acid molecules, the nucleotide sequence that it contains among Fig. 8 to be provided and one or more at the C-of methioninase or the nucleotide sequence of the two or more histidine residues of N-end coding.

28. a discriminating efficiently expresses the method for the transformed host cell of methioninase, this method comprises the following steps:

Under the condition of expressing methioninase, cultivate the host cell of transfection; With

Select peach transformed host cell.

29. the method for claim 28, host cell wherein are escherichia coli (E.coli).

30. the method for claim 29, host wherein cultivates on solid medium.

31. the method for claim 28, wherein said selectivity host produces 5 to 75% the methioninase that is approximately total cell protein.

32. the gene therapy of an improvement, improvement wherein comprises the expression pattern that uses claim 17.

33. the method for claim 32, wherein this expresses the expression of model by tumor-specific promoters control.

34. the method for a purification methioninase, this method comprises the following steps:

A) host of culture expression methioninase under the condition of expressing methioninase;

B) approximately 40-60 ℃ down heating contain the about 1-10 of water-containing buffering liquid minute of extract of the transformant of methioninase;

C) in the GS-3 rotor (Sorval, Du Pont) with about 10k to the extract centrifugalize about 15 minute to 1 hour of 20k rpm after heating;

D) use the filter ultrafiltration supernatant liquid of about 50k to the 100k aperture;

E) under the condition of LIS (approximately 10-50mM) the about pH7.0-7.6 of KCl, carry out the DEAE ion-exchange chromatography, and collect the fraction that contains methioninase with the KCl gradient solution eluting of about 40-200mM;

F) under the condition of ionic medium intensity (approximately 100-200mM) the about pH8.0-8.6 of KCl, carry out the DEAE ion-exchange chromatography second time, and collect the fraction that contains methioninase with the phosphate buffer eluting of 0.1-0.2M KCl;

G) the fraction of in step (e), collecting with can adsorb endotoxic column chromatography medium and contact; With

H) collect the eluent that contains methioninase.

35. the method for claim 34, wherein collected methioninase have the endotoxin that the active of at least 10 units of every milligram of albumen and every milligram of albumen have about 1-100ng.

36. the method for claim 34 is wherein used

post in step (g).

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