CN1300323A - Recombinant (alpha)-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof - Google Patents

Recombinant (alpha)-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof Download PDF

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CN1300323A
CN1300323A CN99806064A CN99806064A CN1300323A CN 1300323 A CN1300323 A CN 1300323A CN 99806064 A CN99806064 A CN 99806064A CN 99806064 A CN99806064 A CN 99806064A CN 1300323 A CN1300323 A CN 1300323A
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iduronidase
enzyme
disease
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E·D·卡基斯
B·塔纳马奇
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HARBOR-UCLA INST OF UNIV OF CALIFORNIA
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Abstract

The present invention provides a recombinant alpha -L-iduronidase and biologically active fragments and mutants thereof, methods to produce and purify this enzyme as well as methods to treat certain genetic disorders including alpha -L-iduronidase deficiency and mucopolysaccharidosis I (MPS I).

Description

Recombinant alpha-L-iduronidase, it is produced and the method for purifying and the method for the treatment of the disease that its shortage causes
Invention field
The present invention relates to molecular biology, zymetology, biological chemistry and clinical medicine domain.Concrete, (method of α-L-iduronidase), production and this enzyme of purifying and treatment comprise that α-L-iduronidase lacks and the method for some inherited diseases of mucopolysaccharidosis I (MPS I) to the invention provides a kind of recombinant alpha-L-iduronidase.
Background of invention
Carbohydrate has played many vital role in the activity of Living Organism.Except their metabolism, carbohydrate is in the human body and other many compositions, as protein and the covalently bound structural constituent of fat (being called glycoconjugate).For example, human connective tissue and cytolemma comprise protein, carbohydrate and proteoglycan matrix.The sugar moieties of this proteoglycan matrix provides important properties to housing construction.
The lysosome that the hereditary defect of the lysosomal enzyme α-L-iduronidase of incision sugar causes being called mucopolysaccharidosis I (MPS I) stores up unusually (Neufeld, E.F., and Muenzer, J. (1989). mucopolysaccharidosis (Scriver, C.R., Beaudet in " inherited disease metabolic basis ", A.L., Sly, W.S. and Valle, D., editor), pp.1565-1587, McGraw-Hill, New York).The severe form of MPS I often is called Hurler syndrome, and and various problems,, coarse facial characteristics fuzzy as mental retardation, cornea, heart trouble, diseases of respiratory system, liver are relevant with splenomegaly, hernia and ankylosis etc.It is just dead before the syndromic patient of trouble Hurler was everlasting 10 years old.The moderate form is called Hurler-Scheie syndrome, the normal not serious moral function that influences, but disease can cause the death more than 10 year old and more than 20 year old.Scheie syndrome is the lightest form of MPS I.It can have ordinary life, but ankylosis, cornea are fuzzy and valvular heart disease causes serious problems.
According to British Columbia investigation, estimate that the sickness rate of MPS I is 1:100 among whole newborn infants, 000 (Lowry etc., human genetics 85:389-390 (1990)), according to Hibernian research, be 1:70,000 (Nelson, human genetics 101:355-358 (1990)).It seems to this disease nobody species diversity.As if this disease of the whole world is all crossed low diagnosis, be not because patient before diagnosis is made just because complication death, exactly because should syndromic gently form can be that sacroiliitis is ignored fully by mistaken diagnosis.Effectively MPS I newborn infant generaI investigation can be found some undiscovered patients in the past.
Except bone marrow transplantation, not to the meaningful treatment of MPS I.Bone marrow transplantation may be effective in the symptom of some these diseases of treatment, but MPS I is had height pathogenicity rate and mortality ratio, and owing to lack suitable contributor, normally infeasible.To and handle this disease to treatment to the feasible another kind treatment of all patients important breakthrough will be provided.
Discovery α-L-iduronidase can be corrected after the enzymatic defect of cultivating the Hurler cell, considers for a long time enzyme is replaced the possible treatment means of treatment as MPS I.In this correction procedure, by receptor-mediated endocytosis, the enzyme that will contain the Man-6-P residue is absorbed into cell, and is delivered to lysosome, and enzyme is removed substrate, Suleparoid and the dermatan sulfate of storing therein.Because the source deficiency of α-L-iduronidase in the tissue, this treatment was also infeasible in the past to people's application.The enzyme replacement notion effectively is used for patient Gaucher with the placenta glucocerebrosidase of modifying first.The transmission of glucocerebrosidase and effective the absorption have proved that enzyme can be absorbed in the body with capacity, provides effective treatment among patient Gaucher.
For α-L-iduronidase treatment of MPS I, the recombinant sources of a kind of enzyme of needs obtains to treat the enzyme supply of quantity sufficient.This mammalian enzyme is cloned (Stoltzfus etc., journal of biological chemistry 267:6570-6575 (1992)) in nineteen ninety, and this people's enzyme was cloned (Moskowitz etc., FASEB J6:A77 (1992)) in 1991.
The accompanying drawing summary
Fig. 1 has represented the Nucleotide of cDNA of coding for alpha-L-iduronidase and the aminoacid sequence of deduction.Nucleotide 1 to 6200 is provided.The amino acid that provides first methionine(Met) from open reading frame to begin.
Fig. 2 representative is according to the result of the elutriant SDS-PAGE of the program acquisition of listing among the embodiment 1.Swimming lane 1 is blank.Swimming lane 2 contains the high molecular standard substance.Swimming lane 3 is blank.Swimming lane 4 contains the bovine serum albumin that concentration is 50 micrograms.Swimming lane 5 to 10 representatives contain the elutriant of the people α-L-iduronidase of 1 microgram, 2 micrograms, 5 micrograms, 5 micrograms, 5 micrograms and 5 microgram amounts reorganization generation respectively.
Fig. 3 has disclosed among 16 MPS patients I the urine GAG level with respect to normal drainage value.In untreated MPS patient I, urine GAG horizontal extent is very wide.After recombinant alpha-L-iduronidase treatment, it is to weigh the effective means that human body responds to treatment more than 50% that the GAG that do not degrade drainage has reduced.
Fig. 4 has shown the white corpuscle iduronidase activity before and after the MPS patient I enzyme treatment.
Fig. 5 has shown oral cavity, enzyme treatment front and back iduronidase activity.
Fig. 6 shown only treated for 8 weeks with recombinase after, among three patients liver and spleen fully dwindle and the joint is reduced relevant more than 65% with the remarkable clinical improvements that soft tissue stores up with the GAG that do not degrade.
Fig. 7 shown only treated for 12 weeks after, in patient with recombinase treatment, the basic normalizing of liver and spleen.
After Fig. 8 had shown that 6 patients treated for 22 weeks with recombinase, urine GAG excretory sharply descended.
The invention summary
On the one hand, feature of the present invention is the method that produces the α-L-iduronidase of its amount treatability use.In a main embodiment, the cDNA that this method comprises coding all or part of α-L-iduronidase is transfected into the step that is fit to its cell of expression.In certain embodiments, used coding complete α-L-iduronidase, the cDNA of preferred people α-L-iduronidase.Yet, in other embodiments, the cDNA of its bioactive fragment of available code or mutant.Particularly, can produce one or more aminoacid replacement, still keep or strengthen the biological activity of enzyme.In other preference, cDNA is transferred to suitable cell or the clone that is used for its expression with expression vector.In a concrete preference, cDNA is transferred to Chinese hamster ovary cell sets up clone 2.131.In other preference, this generating routine has the feature of the extra high generation level of one or more following demonstration: (a) in the production process, the pH of cell growth medium can be low to moderate about 6.5 to 7.0, preferred about 6.7-6.8, (b) per approximately 12 hours interchangeable about 2/3 to 3/4 substratum, (c) with being interrupted the pure oxygen spraying, oxygen saturation the suitableeest about 80%, (d) the initial available microcarrier that contains about 10% serum produces cell lump, transfer to the no protein nutrient solution that is used for producing with getting express developed then, (e) contain one or more compositions of magnitude of recruitment, be selected from L-glutamic acid, aspartic acid, glycine, the no protein of ribonucleoside and dezyribonucleoside or lower protein substratum, as JRH Biosciences PF-CHO product is the suitableeest, (f) in repeatedly batch charging process, and not in the predetermined perfusing course of standard, available perfusion rod as Bellco perfusion rod and (g) Sodium propanecarboxylate of the available gentleness process of inducing induce α-L-iduronidase to express increase.
Aspect second, the invention provides a kind of transfectional cell series, it has the feature of the α-L-iduronidase that can produce its amount treatability use.In preference, feature of the present invention is that a kind of reorganization Chinese hamster ovary line stable and the reliably α-L-iduronidase amount of generation treatability use is as 2.131 clones.In some preferences, clone can contain expression constructs at least about 10 copies.In more preferences, its amount of expression of cell lines is for per 10 7Cell every day is at least about the recombinant alpha-L-iduronidase of 20-40 microgram.
Aspect the 3rd, the invention provides the novel supports of the α-L-iduronidase that is fit to its amount treatability use of generation.In preference, feature of the present invention be comprise cytomegalovirus promoter/enhancer element, the 5 ' intron that constitutes by mouse Ca intron, whole or fragment or the cDNA of mutant and the expression vector in 3 ' Trobest polyadenylation site of coding for alpha-L-iduronidase.In addition, the whole or fragment of optimized encoding α-L-iduronidase or the cDNA of mutant are about 2.2kb.This expression vector can with for example 50 to 1 ratio transfection, strengthen multiple copied and insert with any suitable selection carrier commonly used such as pSV2NEO.In addition, available gene amplification induces multiple copied to insert.
Aspect the 4th, the invention provides the novel α-L-iduronidase that produces according to the inventive method, and its amount treatability is used this enzyme.According to the present invention, every milligram of protein of ratio vigor of α-L-iduronidase is greater than 200,000 units.Preferably, surpass every milligram of about 240,000 units of protein.The molecular weight of α of the present invention-L-iduronidase is about 82,000 dalton, and about 70,000 dalton are amino acid and about 12,000 dalton are carbohydrates.
Aspect the 5th, feature of the present invention is the novel method of purifying α-L-iduronidase.According to first embodiment, cell lump is grown in the substratum that contains 10% serum of having an appointment, forward to then on the no protein production substratum of improvement, produce the height ratio vigor original material that is used for purifying without any remarkable adaptation.Preferably, use to concentrate/the diafiltration flow process, it can remove the required exogenous material of this enzyme of recombinant production, for example, and the tensio-active agent that Pluronics F-68, a kind of protection cell commonly used are not sprayed and damage.Usually these exogenous materials should be separated from a large amount of thick materials, be prevented the post of making dirty.In another preference, the acidifying first post sample solution makes the competitive inhibition effect of the uronic acid of finding in the protein-free culture formulation become minimum.Also be preferred,, but use automatization step and the substratum that can verify to produce pure enzyme with heparin, phenyl and size-exclusion column purification flow process.In another preference, with heparin and phenyl post step remove more undesirable nicked or the degraded α-L-iduronidase.In another preference, come the possible virus of deactivation and do not injure enzyme with the acid pH treatment step.
Aspect the 6th, feature of the present invention is a kind of treatment is lacked the disease that causes wholly or in part by α-L-iduronidase a novel method.In one embodiment, the feature of present method is single with acceptable carrier on recombinant alpha-L-iduronidase or its bioactive fragment or mutant or the coupling pharmacology.In other embodiments, the feature of this method is that the nucleic acid of coding all or part of α-L-iduronidase is transferred in the body in one or more host cells.Preference comprises by the needs of the organism that will treat (preferred mammal or people) optimizes dosage, improves disease symptoms.In preference, disease is mucopolysaccharidosis I (MPS I), Hurler syndrome, Hurler-Scheie syndrome or Scheie syndrome.
Aspect the 7th, feature of the present invention is to be used for the treatment of all or part of novel medicament compositions that comprises α-L-iduronidase that is lacked the disease that causes by α-L-iduronidase.These compositions can be suitable for number of ways to be used, as in parenteral, external application, the nose, suction or Orally administered.In this scope, the feature of embodiment is the nucleotide sequence of coding all or part of α-L-iduronidase, and it can be used in vivo to enter and lacked in the cell that influences by α-L-iduronidase.
Detailed Description Of The Invention
In one aspect, feature of the present invention is to produce the method that its amount treatability is used the α-L-iduronidase of this enzyme.Generally speaking, the feature of present method is the suitable clone of cDNA conversion with coding whole α-L-iduronidase or its bioactive fragment or mutant.Those skilled in the art can prepare except this paper and understand to describe, and are used in those expression constructs in addition with the most desirable generation of the suitable clone of its transfection α-L-iduronidase.In addition, those of skill in the art can be easy to design the bioactive fragment of coding naturally occurring α-L-iduronidase and the cDNA fragment of mutant (they have and the same or analogous biological activity of naturally occurring total length enzyme).
Set up the recombinant sources of α-L-iduronidase, can make up the expression vector of a big series, and test the expression of its α-L-iduronidase cDNA.According to transient transfection experiment and stable transfection, can discern the expression constructs that special high level expression is provided.In one embodiment of the invention, by transfection α-L-iduronidase expression constructs and select to provide the high-expression clone of special high level expression, having developed a kind of Chinese hamster cell is 2.131.According to this embodiment of the invention, such Chinese hamster cell is that comparable normal cell is secretion much about 5,000 to 7,000 times α-L-iduronidase.Therefore, can correctly process the α-L-iduronidase of generation like this, it is absorbed into the cell with high-affinity and correct α-L-iduronidase shortage cell of suffering from Hurler syndrome patient.
The feature that is used to produce the method for α-L-iduronidase that its amount treatability uses is to be a large amount of specially designed production processes of these enzymes that produce.According to the preference of this process, with the cost cheap scale scalable surface of microcarrier as the growth attached cell.
According to the present invention, be used to produce other preference of the method for α-L-iduronidase, optimized a kind of culture systems.In first embodiment, in production process, reduce medium pH to about 6.5 to 7.0, preferably about 6.7-6.8.The advantage of such pH is the gathering that can strengthen lysosomal enzyme more stable under acid pH.In second embodiment, changed about nutrient solution of 2/3 to 3/4 in per 12 hours.An advantage of this program is to strengthen the secretion rate of recombinant alpha-L-iduronidase and gather in the crops more organized enzyme.In the 3rd embodiment,, oxygen saturation is optimized to about 80% with intermittently pure oxygen spraying replacement spraying continuously.In the 4th embodiment, produce a large amount of cells with cytodex 2 microcarriers that contain about 10% serum at first, transfer to the protein-free culture that is used to produce with getting express developed then.In the 5th embodiment, the growth medium as JRH Biosciences PF-CHO product can be optimized to one or more that comprise magnitude of recruitment and be selected from the composition of L-glutamic acid, aspartic acid, glycine, ribonucleoside and dezyribonucleoside.In the 6th embodiment, in repeatedly batch charging process, and not in the predetermined perfusing course of standard, available perfusion rod is as Bellco perfusion rod.In the 7th embodiment, the Sodium propanecarboxylate of the available gentleness process of inducing induces α-L-iduronidase to express to be increased, and does not influence the cell absorption of carbohydrate processing and enzyme substantially.This increase of inducing process that twice can be provided aborning, and significantly do not change the translation post-treatment.
The feature of concrete preference that produces the method for α-L-iduronidase according to the present invention is, one or more or whole optimizing conditions described herein.Therefore production method of the present invention provides the culturing process of the production with following feature:
1. preferably in a large amount of culturing bottles, stir, use cultivation (with Cytodex 2 pearls or its Equivalent) based on microcarrier with top rod (Bellco perfusion rod or its Equivalent).Can in the pH of about 6.7-6.9, cultivate by 10% foetal calf serum in DME/F12 1:1 substratum (with the composition improvement that comprises ribonucleoside, dezyribonucleoside, pyruvic acid, non-essential amino acid and HEPES) and reach the adherent of these pearls.Cultivate in this substratum after 3 days, the beginning flushing process was replaced about 2/3 substratum with protein-free culture in wherein per approximately 12 hours, carried out about 3-4 time altogether and washed.In whole residue incubation time, cell is cultivated in protein-free culture then.
2. preferably culture condition is remained on the dissolved oxygen of 80% air saturation, pH is about 6.7, about 37 ℃ of temperature.This available control tower, operational unit and the proper probes of producing as Wheaton reach.Yet those of skill in the art can understand this easily and can easily be reached by the suitable Controlling System of other manufacturer.Compare with 60% air saturation with 40%, about 80% air saturation causes α-L-iduronidase excretory to improve.Yet 90% air saturation is compared with 80% air saturation does not provide excretory significantly to increase.Can provide dissolved oxygen with 5 microns stainless steel atomizers or its Equivalent by intermittently pure oxygen spraying.About 6.7 pH is best to gathering of α-L-iduronidase.Enzyme is unstable especially at about pH more than 7.0.Below about 6.7 pH, secretion rate can reduce, and particularly is lower than about 6.5 pH.Therefore between about pH of 6.6 to 6.8, kept best cultivation.
3. produce substratum and can be JRH Biosciences and be called the improved form of the patent substratum that the commerce of Excell PF CHO can buy.During with clones such as 2.131 clones, this substratum is supported the secretion level suitable with blood serum medium.Preferably, comprise the acid pH of about 6.7 (+/-0.1) with its improvement, and the HEPES of its available 7.5mM buffering.Substratum can contain 0.05 to 0.1% Pluronics F-68 (BASF), and it is a nonionogenic tenside, or its Equivalent (feature is to have the protection cell to avoid advantage with the relevant shearing force of spraying).Substratum also can contain confirmation and be higher than the existing important patent fill-in of protein-free culture productivity to improving substratum.Those skilled in the art can understand easily, can be according to the selection of optimizing substratum in the feasible concrete commercial embodiment of particular point in time continuously.This variation comprises normal experiment, and will comprise within the scope of the invention.
4. will give up substratum and initial substratum of available amino end acid assay instrument relatively analyzed the production substratum.This analysis has proved that 2.131 clones are depleted to glycine, L-glutamic acid and aspartic acid in the standard P F CHO substratum about 10% level of initial concentration.These amino acid supplementation can be caused strengthening to high level can be than the generation raising 2-3 culture density and the productivity doubly of baseline.Those of skill in the art will understand being used for that other clone in the scope of the invention can be equal and produce α-L-iduronidase according to present method.Therefore, the thing that may need more or less to supplement the nutrients is optimized substratum.These optimizations will be included in the scope of the present invention, and not need over-drastic to test and implement.
5. available core riboside and dezyribonucleoside supplemental medium support to lack the clone 2.131 of Tetrahydrofolate dehydrogenase.Those of skill in the art can understand being used for that other clone in the scope of the invention also can be equal and produce α-L-iduronidase according to present method.Simultaneously may more or less need ribonucleoside and dezyribonucleoside to optimize substratum.These optimizations will be included in the scope of the present invention, and not need over-drastic to test and implement.
6. after cultivation reaches and is paved with 3-4 day, can change about 2/3 nutrient solution in approximately per 12 hours.Can use the replacing of finishing substratum as Bellco perfusion rod (it is a kind of whipping appts, hollow, there is screen filter at the tip).By 40 microns sieve plates on the rod, pump nutrient solution through hollow inside.From the supernatant liquor that contains enzyme, isolate the microcarrier that has 2.131 cell lumps.
7. show that by productivity research the rapid and frequent turnover of substratum can cause the raising of enzyme total collection from cell culture.Lower frequency causes lower total the piling up of enzyme.The enzyme secretion rate be studies have shown that cell is at active Secretases in most of the time at incubation period cultivating in 12 hours in the circulation.More frequent turnover may not necessarily obtain more enzyme.The method of this embodiment has proved that being better than perfusion cultivates, and also is much better than strict batch culture or every day or every other day in batches/reinforced strategy.Use approximately and changed in per 12 hours, cell can be remained on fabulous state with height vigor and high level production rate.
8. can induce the generation of reinforcing alpha-L-iduronidase by the Sodium propanecarboxylate that uses genetic expression.2.131 the systematic study of clone proof can be used about 2mM butyric acid, and causes the enzyme generation that 2 times or bigger inducing are arranged, and minimum to the processing influence of sugar.Also the lower butyric acid level of proof can not induced yet, and higher level can cause higher inducing, but can reduce the avidity of the enzyme of generation to trouble α-patient's that the L-iduronidase lacks cell.Results suggest twice or higher inducing cause sugared less processing and less enzyme to be increased by phosphoric acid addition and toxicity.A concrete preferred method adopts and added the 2mM butyric acid in per 48 hours in culture systems.The enzyme that this embodiment causes using this method to produce the inducing of twice of having an appointment do not have remarkable effect (K-takes in less than 30U/ml or 2.0mM) and enzyme is taken in avidity.Use has above-mentioned all improvement and present method embodiment of inductive feature, and 15 liters of culture systems can produce about every day of 25 milligrams of every liter of nutrient solutions when the peak culture density, or more.
Aspect second, the invention provides the transfectional cell series of unique ability with the α-L-iduronidase that produces its amount treatability use.In preference, feature of the present invention is reorganization such as 2.131 a clones Chinese hamster ovary line, and they can be stablized and produce a large amount of α-L-iduronidase reliably.In preference clone may contain the expression constructs that comprises CMV promotor, C α intron, people α-L-iduronidase cDNA and Trobest polyadenylation sequence at least about 10 copies.In more preferably example, clone with every day per 107 cells reach by correct processing, high absorption form, be suitable for enzyme and replace the α-L-iduronidase for the treatment of at least about the scale of 20-40 microgram.Therefore, for the preference of this aspect of the present invention, be adapted to produce its transfectional cell series of measuring the α-L-iduronidase of treatability use and have one or more following features:
1. the clone of preference is derived from a kind of parental cell line, and wherein cell goes down to posterity through cultivation, up to obtaining littler size and growth velocity and be easy to be attached to substrate up to them faster.
With contain 2 and 3, approximately long people cDNA and the 3 ' Trobest cytomegalovirus promoter/enhancer element of 2.2kb, contain the expression vector transfection preference clone of 5 ' intron of the Ca intron between the mouse exon polyadenylation site.Available as 50 to 1 ratio is with any suitable selection carrier commonly used such as pSV2NEO transfection expression carrier.Select carrier pSV2NEO to give successfully cells transfected conversely with the G418 resistance.In concrete preference, the ratio with about 50 to 1 inserts segmental acquisition because this ratio has strengthened the multiple copied number.According to an embodiment (Chinese hamster ovary line 2.131 wherein is provided), about 10 copies of expression vector of α-L-iduronidase are arranged.Such clone has shown can produce a large amount of people α-L-iduronidase (minimum every day per 10 7 Cell 20 micrograms) activity.Particularly preferred example has to produce correctly as 2.131 clones processes, and contains the ability of the enzyme of the oligosaccharides (contain with the high mannose chain of phosphoric acid 6 modifications, present in an amount at least sufficient to produce the enzyme (K-takes in less than 3nM) with high-affinity) that is connected with N.
3. with being dissolved cell, the elimination mucopolysaccharide stores up and have about 5 days transformation period in patient's cell of suffering from α-L-iduronidase shortage the enzyme quilt that clone of the present invention such as Chinese hamster ovary line 2.131 produce rapidly.
4. the clone of preference adapts to a large amount of the cultivation and the stable under these conditions people of generation α-L-iduronidase as 2.131 clones.The cell of the preference justacrine α-L-iduronidase of growing under about acid pH of 6.6 to 6.8, the accumulation of α under this pH-L-iduronidase can improve.
5. according to the present invention, the concrete preference of clone as 2.131 clones, uses the people α-L-iduronidase of protein-free culture energy twice secretion every day of special preparation above every milliliter 2,000 unit (every milliliter 8 microgram) level.
Aspect the 3rd, the invention provides the novel supports of the α-L-iduronidase that is fit to its amount treatability use of generation.The generation of capacity recombinant alpha-L-iduronidase is the crucial essential condition to enzymatic structure research and enzyme replacement treatment.Allow to produce the recombinant alpha-L-iduronidase of correct processing in order to taking in of a great deal of according to clone of the present invention.Other three kinds of lysosomal enzymes have been described, alpha-galactosidase (Ioannou etc., the cytobiology magazine, 119:1137-1150 (1992)), iduronic acid 2-sulfatase (Bielicki etc., journal of biological chemistry, 289:241-246 (1993)), with N-acetylgalactosamine 4-sulfatase (Anson etc., journal of biological chemistry, 284:789-794 (1992)) in Chinese hamster ovary cell (CHO), use different promoters and come overexpression by amplification in a kind of occasion.Feature of the present invention is the Chinese hamster ovary celI system that lacks Tetrahydrofolate dehydrogenase, but according to preference of the present invention, amplification is unwanted.In addition, the invention provides with CMV immediate early gene promotor/enhanser high level expression people α-L-iduronidase.
The feature of preference of the present invention is the expression vector 5 ' intron that comprises cytomegalovirus promoter/enhancer element, be made of the mouse Ca intron derived from the mouse long-chain immunoglobulin (Ig) C α gene between exon 2 and 3, cDNA and 3 ' the Trobest polyadenylation site that the people is about 2.2kb.Available 50 to 1 ratio and any suitable selection carrier commonly used such as pSV2NEO be this expression vector of transfection together.And select carrier such as pSV2NEO to give successfully cells transfected G418 resistance.In specific embodiment, the expression vector with about 50 to 1 inserts segmental acquisition than selecting carrier because this ratio can strengthen the multiple copied number.According to an embodiment (Chinese hamster ovary line 2.131 wherein is provided), the copy of about 10 α-L-iduronidase expression vector is arranged.Such expression constructs has shown can produce a large amount of people α-L-iduronidase (minimum every day per 10 in suitable clone such as Chinese hamster ovary line 2.131 7 Individual cell 20 micrograms) vigor.
Aspect the 4th, the invention provides the novel α-L-iduronidase that produces according to the inventive method and therefore exist treatability to use the amount of this enzyme.Method of the present invention produces correct processing and be high absorption form, is suitable for that enzyme replaces treatment and to the effective pure substantially α-L-iduronidase of interior therapeutic.
According to the present invention, the ratio vigor of α-L-iduronidase surpasses every milligram of about 200,000 units of protein.Preferably, every milligram of protein surpasses about 240,000 units.The molecular weight of total length α of the present invention-L-iduronidase is about 82,000 dalton, comprises about 70,000 daltonian amino acid and 12,000 daltonian carbohydrates.Recombinase of the present invention is than the partial purification prepared product of the urine enzyme of report can be more effective in the cell endocytosis before.The GAG that effectively reduces radioactivity S-mark according to recombinase of the present invention lacks gathering in the inoblast at α-L-iduronidase, shows that it is transported to lysosome (site that GAG stores).The α of the extremely low concentration that this correction is required-L-iduronidase (half highest correction concentration is 0.7pM), the success of enzyme being replaced treatment is very important.
People α-L-iduronidase cDNA is expected at has 653 amino acid whose protein after signal peptide cuts, and 70,000 daltonian estimated molecular weights are arranged.The L-Ala 26 that amino acid sequencing has disclosed the N-end has provided 629 amino acid whose protein of an expectation.People's recombinant alpha-L-iduronidase has a Histidine in mature protein position 8.The protein sequence of prediction comprises the decorating site that 6 possible N-connect oligosaccharides.All these can be modified in recombinant protein.Proved that the 3rd and the 6th site contain one or more seminose 6-phosphoric acid residues, caused the high-affinity of taking in cell.Following peptide is equivalent to have the amino acid 26-45 of the people's recombinant alpha-L-iduronidase of N-terminal alanine and following sequence:
ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-arg
The overexpression of α of the present invention-L-iduronidase does not cause other to rely on the generally secretion of the lysosomal enzyme of seminose-6-P guiding.Excretory recombinant alpha-L-iduronidase is similar in many aspects with normocrinic enzyme.Find that in various mensuration its molecular weight is 77,82,84 and 89kDa, correct the 87kDa (Barton etc. that the factor is found with urine, journal of biological chemistry, 246:7773-7779 (1971)) and the enzyme 76kDa and the 82kDa (Myerowitz etc. of cultivator fibroblasts to secrete, journal of biological chemistry, 256:3044-3048 (1991); Taylor etc., journal of biological chemistry 274:263-268 (1991)) unanimity.Difference between research neutralization research is because the out of true of measuring.In the born of the same parents of recombinase the graphic-molecular size of processing slowly reduce and the extra appearance less than the 9kDa band at last is identical with human fibroblasts's enzyme.The fastest band is that the incision of 80N-end amino acid proteolysis causes.
Aspect the 5th, feature of the present invention is the novel method of a kind of purifying α-L-iduronidase.In preference, feature of the present invention is the method for a kind of purification of Recombinant α-L-iduronidase, and it has been optimized to provides with the chromatographic resin that can verify and application of sample, washing and wash-out operation easily fast and effective purifying.The method of purifying α of the present invention-L-iduronidase relates to a series of column chromatography steps that can make enzyme purifying of high yield from protein-free production substratum.
According to first embodiment, a large amount of cells are grown in the substratum that contains 10% serum of having an appointment, forward to then on the no protein production substratum of improvement, produce the original material of the height ratio vigor that is used for purifying without any remarkable adaptation.In second embodiment, use to concentrate/the diafiltration flow process, it can remove exogenous material such as Pluronics F-68 prevents that it from polluting post from thick material.In the 3rd embodiment, the acidifying first post sample solution makes the competitive inhibition of the uronic acid found in the protein-free culture formulation etc. become minimum.In the 4th embodiment,, but use the automatization step to produce pure enzyme with heparin, phenyl and size-exclusion column purification flow process.In the 5th embodiment, remove α-L-iduronidase otch or degraded of unwanted generation with heparin and phenyl post step.In the 6th embodiment, come the possible virus of deactivation and do not injure enzyme with the acid pH treatment step.
The feature of the concrete preference of the method for purifying α-L-iduronidase is more than one or all optimizations according to following specific embodiment according to the present invention.Therefore purification process of the present invention can provide the purifying α-L-iduronidase with feature described herein.
1. concentrate/diafiltration: handle thick supernatant liquor with hollow fiber thickener (A/G Technologies, 30K cutoff value), approximately reduce by 75% liquid volume, use heparin sample loading buffer (10mM NaPO then 4, pH5.3, NaCl200mM) diafiltration.Diafiltration is the important step of removing unwanted compound such as Pluronics F-68 (a kind of tensio-active agent that needs, the post of can making dirty) from supernatant liquor many cell cultures of the present invention.Diafiltration also can partly remove to deenergize and hinder the competitive inhibitor that is incorporated into heparin column.These inhibitor can be found in the PF-CHO nutrient solution, and believe that they are derived from the uronic acid that is present in the soybean hydrolyzate in this concrete substratum.
2. heparin column: can be before being added on heparin-SepharoseCL-6B application of sample liquid be adjusted to about 5.0 pH.Other type of heparin column such as heparin FF (Pharmacia) have different keys, discord α-effective combination of L-iduronidase.Lower pH can be to a certain extent in and uronic acid, reduced their competitiveness effect.There are not diafiltration and pH regulator, PF-CHO nutrient solution operation heparin column (not having enzyme stream to wear basically).Available pH is approximately 5.3 damping fluid washing column, uses 0.6M NaCl wash-out then.In conjunction with and the narrow range of wash-out salt concn can draw effective purification step and obtain purity after the step of being everlasting greater than 90% enzyme.
3. phenyl post: can in next step, use phenyl-Sepharose BP (Pharmacia).The heparin elutriant can be adjusted to about 1.5M NaCl, and be added on this post.Resin choice is the same with salt concn important, can guarantee enzyme fully in conjunction with (stream is not worn), and can make things convenient for and wash-out completely with about 0.15M NaCl.The elutriant that obtains is near pure α-L-iduronidase.
4. can carry out the pH deactivation the strong step of removing possible virus is provided.The phenyl amalgamation liquid is adjusted to about 3.3 with citric acid pH3.0, at room temperature kept about 4 hours.Then can in and enzyme.Embodiment with this step has proved the minimum about 5log unit virus of removing of energy.The not deactivation or influence enzymic activity substantially of this step.
5. enzyme can be concentrated and is expelled on the Sephacryl S-200 post then, collect the enzyme peak.
Proved that the enzyme of purifying contains capacity Man-6-P residue in the position 3 and 6 of the sugar of N-connection by this way, provided the enzyme that is lower than every milliliter 30 unit (being lower than 2nM) and take in avidity.This enzyme can fully be corrected mucopolysaccharide and store up disease and have the transformation period in about 5 days born of the same parents.
Aspect the 6th, feature of the present invention is to treat the novel method that is lacked the disease that causes wholly or in part by α-L-iduronidase.Recombinant alpha-L-iduronidase provides dog MPSI model enzyme to replace therapy.This canine model is because transgenation α-L-iduronidase shortage is similar with people MPS I.α-L-iduronidase the vein of purifying, correct processing is applied to 11 dogs.With per kilogram 25,000 to 125,000 units weekly in the dog of dosage treatment 3,6 or 13 months, enzyme is ingested in different tissues, and the lysosome that reduces in many tissues stores up.The long-term treatment of this disease is relevant with the clinical improvements of behavior, ankylosis, epidermis and growth.The more treatment of high dosage (per kilogram 125,000 units weekly) obtains better effect, and except the quicker clinical improvements of behavior, ankylosis, epidermis, also comprises urine GAG excretory normalizing.
Even the low dose of 25,000 units (0.1mg/kg/wk) enzyme treatment also causes significant enzyme to distribute in some tissues and reduces GAG and store up.More than 1 year action, vigor, growth and holistic health situation had significant clinical effectiveness if continue.The treatment of this dosage does not improve other tissue such as cartilage and brain as the significant points of this composition associated diseases.The more high dosage proof of using 5 times 125,000 units (0.5mg/kg) per two weeks can reach the tissue permeability of improvement, and organizes the therapeutic of level to act in 2 weeks and will finish.This increase Research on dose is carried out on two dogs.These MPSI dogs show significant clinical improvements, and the drainage of urine GAG drops in the normal range substantially.Except changing the immune response of medicine-feeding technology control, enzyme treatment does not also show significant clinical or biological chemistry toxicity.It is effectively that this higher enzyme treatment of dosage weekly stores up some Clinical symptoms of improving MPSI and reduction, does not have significant toxicity.
Aspect the 7th, feature of the present invention is to contain people α-L-iduronidase to be used for the treatment of the novel medicament compositions that α-L-iduronidase lacks.This recombinase can be used many approach, as in parenteral, external application, the nose, suction or oral administration use.Others of the present invention provide by with it with can be that acceptable carrier is prepared together and used enzyme on solid, semisolid or liquid or the absorbable capsular pharmacology.The example of pharmaceutical composition comprises tablet, and drops such as nose drops, skin are coated with composition such as ointment, gel, frost and the suspension of usefulness, sucks to use sprays, nasal spray and liposome.Press composition weight, usually recombinase comprises 0.05% to 99% or 0.5% to 99%, contains the composition between 0.5 and 20% and will be used for oral 0.1 to 50% the composition that contains as what will be used to inject.
Produce the pharmaceutical composition of the dosage unit form that contains the therapeutic enzyme that is used for oral administration, can use enzyme and solid, powder carrier, as starch, kelp powder or citrus pulp powder, derivatived cellulose or gelatin such as lactose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, potato starch, W-Gum, amylopectin, also can comprise lubricant such as Magnesium Stearate or calcium, or Carbowax or other polyethylene glycol wax, and be pressed into the core of tablet or drageeing.Drageeing if desired, the available concentrated sugar solution that for example contains gum arabic, talcum and/or titanium dioxide, or the available film that is dissolved in volatile organic solvent or ORGANIC SOLVENT MIXTURES forms agent parcel core.Can in these dressings, add dyestuff, to distinguish the different activities material content.For by gelatin and the soft gelatin capsule composition that for example constitutes as the glycerine of softening agent, or close closed capsule, active substance and Carbowax  or suitable oil can be mixed as sesame oil, sweet oil or peanut oil.Hard gelatin capsule can contain active material particle and solid-state powder carrier such as starch such as lactose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, potato starch, W-Gum or amylopectin, derivatived cellulose or gelatin, but also can comprise Magnesium Stearate or stearic acid as lubricant.
Therapeutic enzyme of the present invention also can pass through the parenteral dispenser, as by subcutaneous, intramuscular or intravenous injection or by the slowly-releasing hypodermic implant.In subcutaneous, intramuscular or intravenous injection, therapeutic enzyme (activeconstituents) can be dissolved or is dispersed in the liquid phase carrier.For the parenteral dispenser, can be with active material and acceptable carrier, preferred peanut oil, the various vegetables oil of cottonseed wet goods suitably mix.Also can use other parenteral carrier as using solketol, the organic composite of glycerine, formal and water-based parenteral formulation.
For using through the injection parenteral, composition can comprise according to the water soluble drug of active acid of the present invention learns the aqueous solution of going up acceptable salt, and it is desirable to concentration is 0.5-10%, can choose the stablizer and/or the buffer substance that comprise in the aqueous solution wantonly.Can be with favourable being potted in the ampoule of the dosage device of solution.
When with the form administering therapeutic enzyme of hypodermic implant, compound is suspended or be dissolved in the slow dispersive material well known by persons skilled in the art, or use with the device of slow release active material by the constant driving of using osmotic pump etc.In these cases, dispenser in can be between one section duration.
For external application, be fit to pharmaceutical composition with ointment, gel, suspension, frost or analogue form.The amount of active substance can change, and for example, active substance weight is between 0.05%-20%.The pharmaceutical composition of this external application can prepare with currently known methods, as with mixing such as active substance and known carrier material such as Virahol, glycerine, paraffin, Stearyl alcohol, polyoxyethylene glycol.Acceptable carrier also can comprise the known chemical absorption enhancer on the pharmacology.The example of absorption enhancer is N,N-DIMETHYLACETAMIDE (U.S. Patent number 3,472,931), ethapon or trifluoroethanol (U.S. Patent number 3,891,757), some alcohol and composition thereof (British Patent No. 1,001,949).Also in british patent specification 1,464, described in 975 the solid support material of the external application dispenser of damaged skin not, it discloses the solid support material that is made of the solvent that comprises 40-70% (v/v) Virahol and 0-60% (v/v) glycerine (as there being any equilibrium composition, being the inert component that is no more than the thinner of solvent cumulative volume 40%).
Containing the dosage that the pharmaceutical composition of therapeutic enzyme uses can change on a large scale, can as severity, the patient age decision of disease, and may be adjusted individually by the various factors.Mention possible range as the therapeutic enzyme amount of dispenser every day and be from about 0.1 milligram to about 2000 milligrams or from 1 milligram to about 2000 milligrams.
May suitable preparation contain the pharmaceutical composition of therapeutic enzyme, thereby make it be provided at dosage in these scopes with single dosage unit or multiple doses unit.Except containing a kind of therapeutic enzyme (or multiple therapeutic enzyme), this prescription also can contain one or more substrate or cofactors of being used for the enzymatic reaction of composition therapeutic.The composition that contains the therapeutic enzyme also can contain more than one therapeutic enzymes.
The recombinase that uses in present method and the composition also can transform patient's cell and use by the nucleic acid with coding recombinant alpha-L-iduronidase.The nucleotide sequence of so encoding can be drawn in the carrier that mixes the cell that is used for being transformed into the individuality that to treat.This paper has described the preference of these carriers.Carrier design can be become to be integrated into this individual karyomit(e), as retrovirus vector, or in host cell self-replicating.The carrier design that comprises the nucleotide sequence of coding for alpha-L-iduronidase can be become can provide continuous or adjustable expression of enzyme.In addition, the genophore of codase can be designed to stable be integrated into cellular genome or only instantaneous existence.The general method of conventional gene therapy can be learned the polynucleotide sequence that is applied to coding for alpha-L-iduronidase.Can be at Friedman, science, 244:1275-1281 (1989); Ledley, J.Inherit.Metab.Dis13:587-616 (1990); With Tolstoshev etc., find the summary of conventional gene therapy technology among the Curr Opinions Biotech1:55-61 (1990).
The concrete preferred method of administered recombinant enzyme is an intravenous administration.Concrete preferred composition comprises recombinant alpha-L-iduronidase, physiological saline, pH is maintained about 5.8 phosphate buffered saline buffer and human albumin.Also can following amount provide these compositions:
α-L-iduronidase 0.05-0.2mg/ml or 12,500-50,000 unit/ml
150mM in the sodium chloride solution IV bag, cumulative volume 50-250cc
Sodium phosphate buffer 10-50mM, pH5.8
Human albumin 1mg/ml
The present invention has been described.The following example explanation subject matter is provided, but only is used for explanation and is not used in restriction.
Embodiment 1
Produce the reorganization iduronidase
Standard technique, for example (1987) " molecular cloning: laboratory manual " 2 such as Sambrook NdEd, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. can be used to the cDNA of clones coding people α-L-iduronidase.With before clone people α-L-iduronidase cDNA from bluescript KS subclone as HindIII-X baI fragment, subclone is gone into PRCCMV (InVitrogen).With clone pRIR14.5 (Kakkis etc., nucleic acids research, 16:7796 (1988)) base 788-1372 (Tucker etc., Proc.Natl.Acad.Sci.USA, the pcr amplification of 78:7684-7688 (1991) has made up the intron box derived from the Cot intron between the exon 2 and 3 of rat immune globulin.This box comprises the 136bp of 3 of exon 2 ' end and 5 ' terminal 242bp of exon 3, and it will be stayed among the cDNA of correct montage.In the coding region of intron box, there is not the ATG sequence.The intron box is cloned into 5 ' HindIII site of α-L-iduronidase cDNA.Remove the neo gene by XhoI digestion, the recirculation carrier produces pCMVhldu then.
Melt a tubule master cell bank, and place it in three T150 bottles, wherein be added with DME/F12 and add fill-in and add 10%FBS and 50011g/mlG418.After 3-4 days, the heavy body that passage to 6 contains same medium is rolled in the bottle with trypsinase-EDTA.With 2 * 10 9The inoculum of cell is added to and contains 60 gram Cytodex2 microcarriers and DME/F12 and add fill-in, 10%FBS and 50011g/ml G418's, final volume is in 13 liters the Wheaton microcarrier bottle.Pass through Bellco top drive blender jar with perfusion rod agitator.By temperature, DO and pH monitoring are cultivated, and control with the small-sized pilot plant Controlling System of the Wheaton with PC interface (BioPro software).With the parameter heating jacket, oxygen atomizer and alkali pump are controlled at setting point: 37 ℃, and 80% air saturation and pH6.7.Culture is cultivated 3-4 day, and culture is by 1-3 * 10 at this moment 6The logarithmic growth of cell/ml produces.After this, every 12 hours, (, JRHBiosciences) replace substratum once according to the customer requirement improvement with the PF-CHO substratum.2 parts of gleanings are placed on one side as " washings ", and the 3rd part of gleanings is the effusive beginning of product.Added the increase that conclusive 2mM Sodium propanecarboxylate induces iduronidase to express in per 48 hours.Replaced 10 liters of substratum in per 12 hours and continue to produce, and 1 micron filter membrane of gleanings filtration is removed free cell and fragment.The temperature of continuous monitoring culture, pH and DO.Twice of every day to the culture sampling, and with phase-contrast microscopy cell state and microorganism, tested glucose content with portable glucose meter, usefulness fluorogenic substrate test check iduronidase activity before substratum replaces it.Check cell concentration for several times at the volley with the test of total cell protein matter.Be in operation on the way, cell concentration reaches 10 7Cell/ml.The production culture that contains iduronidase that to collect then concentrates 5 times with A/GTechnology hollow fiber molecule membrane retention value molecular weight 30,000.Then with the 10mMNaPO of concentrated solution with at least 3 times of volumes 4, the 0.2M NaCl diafiltration among the pH5.8 8 hours.This step is removed Pluronics F68 and uronic acid from concentrated solution.These molecules can suppress the function of heparin column.Concentrated solution is adjusted to pH5.0, filters 1.0 and 0.2 microns filter membranes, be added to then on heparin-Sepharose CL-6B post.With the 0.2M NaCl of 10 times of column volumes, 10mM NaPO 4, the pH5.3 washing column is used 0.6MI, 10mM NaPO 4, pH5.8 wash-out enzyme.Elutriant is adjusted to 1.5M NaCl, filters 1 micron filter membrane, and be added on phenyl-Sepharose HP post.With the 1.5M NaCl of 10 times of column volumes, 10mM NaPO 4, the pH5.8 washing column, and use 0.15M NaCl, 10mMNaPO 4, pH5.8 wash-out enzyme.
Partly carry out inactivation of virus to pH3.3 with 1M citric acid pH2.9 acidifying enzyme, and in room temperature, pH3.3 is insulation enzyme 4 hours down, with the 1M phosphate buffered saline buffer with the enzyme re-adjustment to pH5.8.This step of proof can be removed 5log or more retrovirus in the experiment of spiking.Inactivator is filtered 0.2 μ filter membrane, upward concentrate, be expelled in the circulation of SephacrylS200 gel-filtration column, and collect the peak at A/G Technologies hollow fiber concentrating instrument (cutoff value molecular weight 30,000).The peak that merges is filtered 0.2 μ filter membrane, be made into 0.1MNaPO 4, the pH5.8 and the test tube of packing into.
Can carry out a series of research comes quality, the purity of tested enzyme and tires.The SDS-PAGE result of eluate is provided in Fig. 2.
A kind of recombinant human alpha-L-the iduronidase that obtains from this program has shown tiring of every milliliter 100,000 unit, and has total protein concentration 0.313mg/ml.
Embodiment 2
Recombinant alpha-L-iduronidase treatment is effective
9 MPSI dogs and 6 MPSI cats with the recombinant alpha-L-iduronidase short-term intravenously administrable of purifying, have been shown that enzyme is significantly taken in various tissues, and behind single administration, estimated at 50% or be absorbed more in 24 hours in the tissue.Though liver and spleen have absorbed the enzyme of volume, and have best pathology to improve, many be not the improvement of all having observed pathology and mucopolysaccharide content in the tissue.Specifically, cartilage, brain and heart valve significantly do not improve.Observe the clinical improvements of a dog in 13 months long-term treatment, but other research was limited in 6 months or is following.Accept all dogs of recombinant human enzyme and most of cat and antibody occurred people's product.IgG antibody is complement activation type (may be dog IgG Equivalent).Also in the sick patient of at least 13% Gaucher that treats with glucocerebrosidase, observed this phenomenon.Also having observed in a dog may the albuminuria relevant with immune-complex disease (ICD).In other treatment animal, do not observe other antibody effect.Do not observe special toxicity, clinical labororatory's research (complete blood count, ionogen, BLJN/ creatinine, liver enzyme, urinalysis) is also normal in addition.
Promptly use the enzyme treatment of low dose of 25,000 units (0.1mg/kg/wk) also to cause having significant enzyme to distribute in some tissues, and reduce GAG and store up.If continue more than 1 year, the remarkable clinical effectiveness about behavior, vigor, growth and holistic health of treatment is tangible.The treatment of this dosage does not improve other tissue such as the cartilage and the brain at this important pathogenic position of composition.The more high dosage proof of using 5 times 125,000 units (0.5mg/kg) per two weeks can reach the tissue permeability of improvement, and organizes the therapeutic of level to act in 2 weeks and just can finish.6 months by a definite date research is just carried out on two dogs to this increase dosage.These MPSI dogs show significant clinical improvements, and the drainage of urine GAG drops in the normal range substantially.Except the immune response that changes application technique control, enzyme treatment does not also show significant clinical or biological chemistry toxicity.The enzyme treatment of weekly more high dosage is to some Clinical symptoms of improving MPSI and reduce that to store up be effectively, and does not have significant toxicity.
These are to each research of MPSI dog with to the research of MPSI cat, and the result has shown that recombinant human alpha-L-iduronidase is safe.These identical results also provide important basis: it is effective that this recombinase lacks treatment α-L-iduronidase.
Embodiment 3
To the effective recombinant alpha of people-L-iduronidase treatment
The people cDNA of α-L-iduronidase has predicted after the signal peptide cutting to be had 653 amino acid whose protein and 70,000 daltonian estimated molecular weights is arranged.The L-Ala 26 that amino acid sequencing has disclosed the N-end has provided 629 amino acid whose expectation protein.People's recombinant alpha-L-iduronidase has the Histidine of mature protein position 8.The protein sequence of this prediction comprises the oligosaccharides decorating site that 6 possible N-connect.All these sites are modified in recombinant protein.Shown that the 3rd and the 6th site contains one or more seminose 6-phosphoric acid residues, they can cause that high-affinity takes in cell.
This peptide is equivalent to the amino acid 26-45 of people's recombinant alpha-L-iduronidase, has N-terminal alanine and following sequence:
ala-glu-ala-pro-his-leu-val-his-val-asp-ala-ala-arg-ala-leu-trp-pro-leu-arg-arg
Recombinase has 82,000 daltonian apparent molecular weights because carbohydrate is modified on SDS-PAGE.By UCLA protein sequencing equipment purifying people recombinant alpha-L-iduronidase is checked order.Preferred intravenous administration recombinase.People's recombinant alpha-L-iduronidase with 0.05-0.2mg/ml (12,500-50, the concentration of 000 unit/mL) is with the supply of 10mL polypropylene bottle.The final dose form of enzyme comprises people's recombinant alpha-L-iduronidase, physiological saline, the phosphate buffered saline buffer of pH5.8 and the human albumin of 1mg/ml.These can prepare in normal saline bag.
Components composition
α-L-iduronidase 0.05-0.2mg/mL or 12,500-50,000 unit/mL
150mM in the sodium chloride solution IV bag, cumulative volume 50-250cc
Sodium phosphate buffer 10-50mM, pH5.8
Human albumin 1mg/mL
Comprised the clinical phenotypes that shows the MPSI disease under study for action, and the patient of α in white corpuscle and the inoblast-L-iduronidase level subnormal 1%.All patients show that mucopolysaccharide gathers and some clinical evidences of function damage in various degree in internal organ and soft tissue.Drain the per-cent that reduces in time by measurement urine GAG and can determine effect.Fig. 3 has disclosed 16 urine GAG levels that patient MPSI compares with normal drainage value.In untreated patient MPSI, urine GAG value wide range.After recombinant alpha-L-iduronidase treatment, the GAG that do not degrade drains and descends more than 50%, is a kind of effective means of measuring individuality to the reaction of treatment.Fig. 4 has shown the activity of enzyme treatment front and back white corpuscle iduronidase among patient MPSI.The oral cavity iduronidase activity of enzyme treatment front and back has been described in Fig. 5.Fig. 6 has shown the contraction greatly of liver and spleen among three patients, and the remarkable clinical improvements that stores up of joint and soft tissue, and is relevant greater than 65% minimizing with the recombinase treatment GAG that only do not degrade after 8 weeks.Fig. 7 has shown with recombinase treatment only after 12 weeks, with the basic normalizing of liver and spleen among the patient of recombinase treatment.Fig. 8 has shown that urinating the GAG excretory with 22 weeks of recombinase treatment among 11 patients sharply descends.The clinical assessment that liver and splenomegaly are little is the method for accepting extensively the most (Hoogerbrugge etc., Lancet345:1398 (1995)) that bone marrow transplantation successful among assessment patient MPSI is treated.These are measured in patient MPSI and reduce internal organ GAG and store up height correlation.
Though the present invention is described according to existing preference, it must be understood that, but produce various changes, and do not surmount the scope of spirit of the present invention.Therefore the present invention is limited by claim only.

Claims (41)

1. a method that produces α-L-iduronidase is characterized in that, the cDNA that this method comprises with coding total length α-L-iduronidase or its bioactive fragment or mutant transforms suitable clone.
2. the method for claim 1 is characterized in that, suitable clone is Chinese hamster ovary line 2.131.
3. method as claimed in claim 2, it is characterized in that, Chinese hamster ovary cell secretion in this method approximately Duos 5 than excretory α-L-iduronidase before the cDNA that introduces coding total length α-L-iduronidase or its bioactive fragment, 000 to 7,000 times α-L-iduronidase.
4. the method for claim 1 is characterized in that, transfectional cell is grown on the microcarrier.
5. the method for claim 1 is characterized in that, culture systems is optimized to the cultivation pH that makes in the production process is reduced to about 6.7-6.8.
6. the method for claim 1 is characterized in that, about 2/3 to 3/4 replacing in per approximately 12 hours of culture systems growth medium once.
7. the method for claim 1 is characterized in that, the culture systems oxygen saturation is optimized to about 80%.
8. method as claimed in claim 7 is characterized in that, with pure oxygen spray method intermittently the culture systems oxygen saturation is optimized to about 80%.
9. the method for claim 1 is characterized in that, produces a large amount of cells that are used for culture systems with the microcarrier that contains about 10% serum at first.
10. the method for claim 1 is characterized in that, this method also comprises flushing and transfers to the protein-free culture that is used to produce.
11. the method for claim 1 is characterized in that, has used the culture systems that comprises JRH BiosciencesPF-CHO growth medium.
12. method as claimed in claim 11 is characterized in that, optimizes described growth medium and is one or more that comprise magnitude of recruitment and be selected from the composition of L-glutamic acid, aspartic acid, glycine, ribonucleoside and dezyribonucleoside.
13. the method for claim 1 is characterized in that, carries out the batch charging process with the perfusion rod.
14. the method for claim 1 is characterized in that, Sodium propanecarboxylate is added in the culture systems.
15. the transfectional cell series of generation α-L-iduronidase ability is arranged.
16. transfectional cell series as claimed in claim 15 is characterized in that, transfectional cell series is a kind of reorganization Chinese hamster ovary line.
17. transfectional cell series as claimed in claim 15 is characterized in that, this transfectional cell series is reorganization Chinese hamster ovary 2.131 clones.
18. transfectional cell series as claimed in claim 15, it is characterized in that, this transfectional cell series contains at least 10 expression constructs copies that comprise CMV promotor, C α intron, α-L-iduronidase cDNA and Trobest polyadenylation sequence.
19. transfectional cell series as claimed in claim 15 is characterized in that, this transfectional cell series expression amount is every day per 10 at least 7The α of the about 20-40 microgram of cell expressing-L-iduronidase.
20. an adaptation produces the carrier of people α-L-iduronidase in transfectional cell.
21. carrier as claimed in claim 20 is characterized in that, this carrier adapts to generation people α-L-iduronidase in Chinese hamster ovary cell CHO.
22. carrier as claimed in claim 20 is characterized in that, this carrier comprises a CMV immediate early gene promotor/enhanser.
23. carrier as claimed in claim 20, it is characterized in that, this carrier comprises cytomegalovirus promoter/enhancer element, by 5 ' intron that the mouse Ca intron between exon 2 and 3 constitutes, cDNA and 3 ' Trobest polyadenylation site of coding total length α-L-iduronidase or its bioactive fragment.
24. recombinant alpha-L-iduronidase that method according to claim 1 produces.
25. α-L-iduronidase that method according to claim 1 produces is characterized in that this enzyme has about at least every milligram 200,000 unit and compares vigor.
26. α as claimed in claim 25-L-tilactase is characterized in that, this enzyme has the ratio vigor of about at least every milligram 240,000 unit.
27. the method for purifying α-L-iduronidase is characterized in that this method comprises the following steps:
(a) concentrate/the diafiltration program removes the compound that one or more are not wanted from sample;
(b) sample of acidification step (a);
(c) sample of step (b) is flow through from heparin column;
(d) sample of step (c) is flow through from the phenyl post;
(e) sample of step (d) is flow through from the Sephacryl post; With
(f) α-L-iduronidase of basic purifying is flow through.
28. all or part of method by α-disease that L-iduronidase shortage causes of treatment is characterized in that this method comprises the step of administered recombinant α-L-iduronidase.
29. all or part of method by α-human disease that L-iduronidase shortage causes of treatment is characterized in that this method comprises the step of administered recombinant people α-L-iduronidase.
30. method as claimed in claim 28 is characterized in that, this disease is a mucopolysaccharidosis.
31. method as claimed in claim 28 is characterized in that, this disease is MPSI.
32. method as claimed in claim 28 is characterized in that, this disease is selected from Hurler disease, Scheie syndrome and Hurler-Scheie syndrome.
33. method as claimed in claim 28 is characterized in that, suffer from the patient's of this disease α-L-iduronidase active for normal activity about 1% or lower.
34. method as claimed in claim 28 is characterized in that, is applied once in a week to the patient who suffers from its deficiency disease at least about 25,000 units or 0.1mg/kg recombinant alpha-L-iduronidase.
35. method as claimed in claim 28 is characterized in that, is applied once in a week to the patient who suffers from its deficiency disease at least about 125,000 units or 0.5mg/kg recombinant alpha-L one iduronidase.
36. a pharmaceutical composition is characterized in that, said composition comprises acceptable carrier on recombinant alpha-L-iduronidase and the pharmacology.
37. pharmaceutical composition as claimed in claim 36 is characterized in that, said composition also comprises sodium chloride solution, damping fluid and human albumin.
38. pharmaceutical composition as claimed in claim 36 is characterized in that, this recombinant alpha-L-iduronidase is approximately 0.05 with concentration and exists to about 50,000 units to 0.20mg/mL or every milliliter about 12,500.
39. pharmaceutical composition as claimed in claim 36 is characterized in that, described human albumin exists with the concentration of about at least 1mg/mL.
40. pharmaceutical composition as claimed in claim 36 is characterized in that, described damping fluid is that concentration is the sodium phosphate buffer of about 10-50mM.
41. pharmaceutical composition as claimed in claim 36 is characterized in that, the pH of described composition maintains about 5.8.
CN99806064A 1998-05-13 1999-05-07 Recombinant (alpha)-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof Pending CN1300323A (en)

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CN107699590A (en) * 2017-10-13 2018-02-16 成都中医药大学 A kind of method of Prepare restructuring people α L iduronases
CN114181318A (en) * 2021-11-08 2022-03-15 四川大学 Recombinant adeno-associated virus for expressing IDUA fusion protein penetrating blood brain barrier in high-efficiency tissue specificity and application

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US6569661B1 (en) * 1999-11-12 2003-05-27 Biomarin Pharmaceutical Inc. Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating diseases caused by deficiencies thereof
US6585971B1 (en) * 1999-11-12 2003-07-01 Harbor-Ucla Research And Education Institute Recombinant α-L-iduronidase, methods for producing and purifying the same and methods for treating disease caused by deficiencies thereof
EP3449934B1 (en) 2000-07-18 2020-05-20 Duke University Treatment of glycogen storage disease type ii
US7442372B2 (en) * 2003-08-29 2008-10-28 Biomarin Pharmaceutical Inc. Delivery of therapeutic compounds to the brain and other tissues
US9222088B2 (en) * 2010-10-22 2015-12-29 Curna, Inc. Treatment of alpha-L-iduronidase (IDUA) related diseases by inhibition of natural antisense transcript to IDUA
JP6591956B2 (en) * 2013-03-15 2019-10-16 ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア Compositions and methods for treating MPS1
AU2017215211C1 (en) 2016-02-03 2023-11-16 The Trustees Of The University Of Pennsylvania Gene therapy for treating mucopolysaccharidosis type i
WO2018144441A1 (en) * 2017-01-31 2018-08-09 Regenxbio Inc. Treatment of mucopolysaccharidosis i with fully-human glycosylated human alpha-l-iduronidase (idua)
SG11201912631PA (en) 2017-07-06 2020-01-30 Univ Pennsylvania Aav9-mediated gene therapy for treating mucopolysaccharidosis type i
CN115109790A (en) * 2021-03-23 2022-09-27 北京据德医药科技有限公司 Recombinant a-L-iduronate prase and preparation method thereof

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ATE426031T1 (en) * 1995-09-14 2009-04-15 Virginia Tech Intell Prop PRODUCTION OF LYSOSOMAL ENZYMES IN PLANT EXPRESSION SYSTEMS

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CN103487414A (en) * 2012-06-14 2014-01-01 北京和信非凡生物技术有限公司 Method, substrate and reagents for alpha-L-iduronidase activity detection
CN107699590A (en) * 2017-10-13 2018-02-16 成都中医药大学 A kind of method of Prepare restructuring people α L iduronases
CN114181318A (en) * 2021-11-08 2022-03-15 四川大学 Recombinant adeno-associated virus for expressing IDUA fusion protein penetrating blood brain barrier in high-efficiency tissue specificity and application

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