WO2011108451A1 - Procédé de production d'enzymes lysosomales recombinantes au moyen de cellules présentant une inactivation de gène - Google Patents

Procédé de production d'enzymes lysosomales recombinantes au moyen de cellules présentant une inactivation de gène Download PDF

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WO2011108451A1
WO2011108451A1 PCT/JP2011/054282 JP2011054282W WO2011108451A1 WO 2011108451 A1 WO2011108451 A1 WO 2011108451A1 JP 2011054282 W JP2011054282 W JP 2011054282W WO 2011108451 A1 WO2011108451 A1 WO 2011108451A1
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gene
lysosomal enzyme
cells
sulfatase
enzyme
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PCT/JP2011/054282
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Japanese (ja)
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厚 杉村
八重 伊東
啓之 薗田
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日本ケミカルリサーチ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)

Definitions

  • the present invention relates to a method for producing a recombinant lysosomal enzyme using a gene knockout cell.
  • the recombinant lysosomal enzyme is an endogenous gene encoding a lysosomal enzyme such as galactosidase A or iduronic acid 2-sulfatase.
  • the present invention relates to a method for producing a gene using a knockout cell having a disrupted gene.
  • Lysosomal enzymes are a group of hydrolases having an optimum pH in an acidic region localized in lysosomes, such as ⁇ -L-iduronidase, iduronic acid 2-sulfatase, galsulfase (N-acetylgalactosamine-4-sulfate sulfatase), acidic Enzymes such as ⁇ -glucosidase, ⁇ -galactosidase A, glucocerebrosidase, sulfamidase are included in this category.
  • Lysosomal disease is a general term for diseases caused by genetic deficiency of lysosomal enzymes, and this category includes diseases such as lipid accumulation disease, mucopolysaccharidosis, and glycoprotein metabolism disorders.
  • ⁇ -L-iduronidase gene deficiency is Harler's syndrome or Cheyre's syndrome
  • iduronic acid 2-sulfatase gene deficiency is Hunter syndrome
  • galsulfase gene deficiency is Maroto Lamy syndrome
  • acid ⁇ -glucosidase gene deficiency is Pompe disease
  • the ⁇ -galactosidase A gene deficiency causes Fabry disease
  • the glucocerebrosidase gene deficiency causes Gaucher disease (see Non-Patent Document 1).
  • enzyme replacement therapy is carried out in which an enzyme that is deleted or decreased in a patient is supplemented from outside the body by intravenous injection or the like.
  • Recombinant enzymes used for treatment are produced using mammalian cells such as CHO cells transformed with an expression vector incorporating a gene encoding the enzyme.
  • the lysosomal enzyme is originally an enzyme localized in the lysosome, but it is secreted to the outside by forced expression. Therefore, by culturing transformed cells, the secreted enzyme can be obtained in the culture solution. it can.
  • a method for producing recombinant lysosomal enzyme by expressing it in mammalian cells for example, a method for producing recombinant ⁇ -L-iduronidase using CHO cells has been reported (see Patent Document 1). .
  • Patent Document 2 There is also a report on a method for producing recombinant iduronic acid 2-sulfatase using CHO cells.
  • recombinant galsulfase In order to use a recombinant enzyme as a medicine, it is necessary to highly purify the expressed enzyme. For example, a method for highly purifying recombinant galsulfase has been reported for the purpose of obtaining a pharmaceutical formulation (see Patent Document 5). Here, recombinant galsulfase is purified to a purity of 99.9% or more. However, in order to use it as a pharmaceutical product, it is necessary to eliminate contamination even if it is a trace amount in the case of harmful impurities. . In particular, in the case of a lysosomal enzyme used in enzyme replacement therapy, administration to a patient is continued. Therefore, if a foreign substance, such as a protein derived from an endogenous gene of a CHO cell, is mixed, an antigen-antibody reaction occurs and the patient Serious side effects such as allergic symptoms may occur.
  • a foreign substance such as a protein derived from an endogenous gene of a
  • the recombinant lysosomal enzyme When the recombinant lysosomal enzyme is produced using mammalian cells, it is expected that the lysosomal enzyme derived from the endogenous gene of the host cell will remain actively in the lysosome without being secreted outside the cell. Therefore, it is considered that the possibility that the lysosomal enzyme derived from the endogenous gene of the host cell is mixed with the recombinant lysosomal enzyme is low. For this reason, as far as the present inventors know, there is no report on a method for producing a recombinant lysosomal enzyme provided with a step for removing a lysosomal enzyme derived from an endogenous gene of a host cell. In addition, there is no report analyzing whether or not a recombinant lysosomal enzyme is mixed with a lysosomal enzyme derived from an endogenous gene of a host cell during the production process.
  • an object of the present invention is a method for producing a recombinant lysosomal enzyme produced using mammalian cells, which can also prevent contamination of trace amounts of lysosomal enzymes derived from endogenous genes of the mammalian cells. It is to provide a manufacturing method.
  • the inventors of the present invention have attempted to produce a recombinant lysosomal enzyme using a knockout cell in which the gene of lysosomal enzyme is disrupted, and found that the above object can be achieved.
  • the present invention has been completed based on this discovery.
  • a method for producing a recombinant human lysosomal enzyme comprising the following steps: (A) incorporating a gene encoding a human lysosomal enzyme into an expression vector so as to be expressed in a mammalian cell; (B) introducing the expression vector into a mammalian cell in which an endogenous gene encoding one or more lysosomal enzymes has been disrupted to produce a human lysosomal enzyme gene expression cell; and (c) the human lysosome.
  • a method comprising (2) the endogenous gene encoding the lysosomal enzyme is selected from ⁇ -galactosidase A, iduronic acid 2-sulfatase, ⁇ -L-iduronidase, galsulfase, acid glucosidase, glucocerebrosidase, sulfamidase, 1 or The method according to (1) above, which is a plurality of genes.
  • the endogenous gene encoding the lysosomal enzyme is one or more genes selected from ⁇ -galactosidase A, iduronic acid 2-sulfatase, and sulfamidase.
  • the gene encoding the human lysosomal enzyme is selected from ⁇ -galactosidase A, iduronic acid 2-sulfatase, ⁇ -L-iduronidase, galsulfase, acid glucosidase, glucocerebrosidase, (1) or (2 )the method of.
  • disrupted endogenous gene is a gene encoding the same type of lysosomal enzyme corresponding to the introduced human lysosomal gene.
  • the introduced human lysosomal gene is ⁇ -galactosidase A or iduronic acid 2-sulfatase.
  • a lysosomal enzyme derived from an endogenous gene of a mammalian cell from being mixed into a recombinant human lysosomal enzyme obtained by expression in a mammalian cell (FIG. 2).
  • the analysis result by RP-HPLC method of recombinant human iduronic acid 2-sulfatase purified product is shown.
  • the characteristics of the production method of recombinant human iduronic acid 2-sulfatase using gene knockout cells are schematically shown.
  • (A) shows the case where cells that do not disrupt the gene are used, and
  • (B) shows the case where gene knockout cells are used according to the present invention.
  • the lysosomal enzyme refers to a hydrolase present in the lysosome, particularly an enzyme that hydrolyzes a polysaccharide.
  • ⁇ -galactosidase A iduronic acid 2-sulfatase, ⁇ -L-iduronidase, galsulfase, Contains enzymes such as acid glucosidase, glucocerebrosidase, and sulfamidase.
  • the term “recombinant human lysosomal enzyme” refers to a human lysosomal enzyme produced using a gene recombination technique. In general, it is produced by incorporating a gene encoding human lysosomal enzyme into an expression vector, transforming a mammalian cell using the gene, and culturing the transformed cell.
  • a gene encoding human lysosomal enzyme into an expression vector, transforming a mammalian cell using the gene, and culturing the transformed cell.
  • cDNA including the entire translation region of the gene encoding the natural enzyme including the leader peptide region is preferably used.
  • Other genes such as those of growth hormone, erythropoietin, or other lysosomal enzymes may be used, and the amino acid sequence of the native enzyme is not altered in the translated region of the cDNA It is also possible to use a gene having such a base substitution, or a gene having a base substitution, deletion or duplication so as to bring about substitution, deletion or duplication of the amino acid sequence of the natural enzyme in the translation region of cDNA.
  • the expression vector is not particularly limited as long as it can express a gene encoding a human lysosomal enzyme in a mammalian cell, and is generally in the form of a plasmid cut by a circular or restriction enzyme. Introduce into cells.
  • a gene encoding a human lysosomal enzyme is generally incorporated downstream of a promoter that controls the expression of the gene so as to be expressed in mammalian cells.
  • a promoter derived from cytomegalovirus (CMV), an SV40 early promoter, an elongation factor 1 (EF-1) promoter, or the like can be used.
  • mammalian cells are not particularly limited as long as they can introduce an expression vector to express a human lysosomal enzyme, but CHO cells and COS cells can be preferably used.
  • an endogenous gene refers to a gene that a mammalian cell originally has on its genome. If the mammalian cell is a CHO cell, it is a Chinese hamster gene, and if it is a COS cell, it is African. It is a gene of midisal. Endogenous genes encoding lysosomal enzymes include ⁇ -galactosidase A, iduronic acid 2-sulfatase, ⁇ -L-iduronidase, galsulfase, acid glucosidase, glucocerebrosidase, sulfamidase. In normal cells, genes encoding various lysosomal enzymes are expressed and localized in lysosomes.
  • a gene knockout cell mainly refers to a cell in which both of a pair of genes existing on a homologous chromosome are destroyed so that proteins encoded by those genes are not expressed at all.
  • Gene knockout cells can be constructed in vitro from desired cells by homologous recombination (Huang DC. Et. Al., Mol. Cancer Res. (2002) 1, 56-57).
  • Adeno-associated virus vectors for generating gene knockout cells have also been developed, and methods for producing gene knockout cells in vitro are well known to those skilled in the art (Porteus MH. Et. Al., Mol Cell Biol (2003) 23, 3558-65).
  • genes present on the X chromosome include iduronic acid 2-sulfatase and ⁇ -galactosidase A genes.
  • Gene knockout cells can also be obtained by establishing a knockout animal cell line.
  • Knockout animals with disrupted genes encoding lysosomal enzymes include, for example, knockout mice of the iduronic acid 2-sulfatase gene (Garcia (AR. Et al., J Inherit Metab Dis. (2007) 30, 924-34), ⁇ -galactosidase A Gene knockout mice (Ioannou YA. Et al., Am J Hum Genet. (2001) 68, 14-25) are known, and gene knockout cells of lysosomal enzymes can be obtained by establishing cells from these animals. Obtainable.
  • siRNA small interfering RNA
  • siRNA can be introduced into a cell by transforming the cell with an siRNA expression vector.
  • siRNA expression vectors such as pBAsi series, pSINsi series, and pSINsi-DK series (Takara Bio). For example, these commercially available products were used to transform with siRNA expression vectors. Gene knockout mouse cells can be easily created.
  • a gene knockout cell used for producing a recombinant human lysosomal enzyme is a cell in which one or a plurality of endogenous lysosomal genes are disrupted, and particularly corresponds to a recombinant human lysosomal enzyme.
  • a cell in which a gene encoding the same kind of enzyme is disrupted is most preferably used.
  • [Construction of recombinant human iduronic acid 2-sulfatase expression vector] A cDNA containing the entire translation region of human iduronic acid 2-sulfatase (the DNA sequence is shown in SEQ ID NO: 1 and the amino acid sequence encoded by it is shown in SEQ ID NO: 2. The first 25 amino acids are the leader peptide).
  • the expression vector (2 ⁇ g) was transfected and introduced into 1 ⁇ 10 7 CHO cells (CHO-K1 system: purchased from RIKEN) using Lipofectamine 2000 (Invitrogen), and further introduced. , After selective culture in Ham-F12 medium (Invitrogen) containing 0.8% / mL of 10% fetal bovine serum (FBS) and G418 (SIGMA) to obtain transformed cells that have acquired neomycin resistance Stable, high-producing strains were selected by cell cloning.
  • FBS fetal bovine serum
  • SIGMA G418
  • EX-CELL302 medium JRH
  • 4 mM L-glutamine 4 mM L-glutamine
  • 10 mg / L hypoxanthine 4 mg / L thymidine
  • 120 mg / L G418 120 mg / L G418
  • Pre-culture of transformed cells for expression of recombinant human iduronic acid 2-sulfatase The seed cells were diluted to a concentration of 2 ⁇ 10 5 cells / mL, and L-glutamine 4 mM, hypoxanthine 10 mg / L, thymidine 4 mg / L, and G418 120 mg / L were added to EX-CELL302 medium (JRH). And at 37 ° C. in the presence of 5% CO 2 and passaged every 3-5 days until 4 ⁇ 10 10 cells were obtained.
  • the cells obtained by the above pre-culture were transferred to a 200 L jar fermenter (Able) at a cell concentration of about 2 ⁇ 10 5 cells / mL, and 5% CO 2 at 37 ° C. with stirring at 100 rpm. Cultured in the presence of 2 . Aeration was carried out by upper surface and in-liquid aeration, and the dissolved oxygen amount was set to 70%. During the cultivation, the glucose concentration in the medium was monitored, and when the glucose concentration became 9.5 mmol / L or less, a 0.3 g / L glucose solution was added so that the final concentration was 19 mmol / L.
  • EX-CELL302 medium supplemented with 4 mM L-glutamine and 10 mg / L insulin was added.
  • the main culture was terminated 9 days after the start of the culture.
  • a column (column volume; about 6.3 L) was loaded and adsorbed at a linear flow rate of 150 cm / hour. Subsequently, the column was washed with 50 mM sodium acetate / 150 mM NaCl (pH 4.3) solution in 4 times the column volume at the same flow rate, and then 50 mM sodium acetate / 150 mM NaCl (pH 5.1) solution in 5 times the column volume. The eluted protein was eluted with
  • TNBP tri-n-butyl phosphate
  • captoMMC column elution fraction were added to the final concentrations of 0.3% (v / v) and 1% (w / v), respectively.
  • Tween 80 was added and allowed to stand at room temperature for 6 hours.
  • the pH was adjusted to 7.0 by adding 6N NaOH to the elution fraction of the CaptoMMC column subjected to the virus inactivation treatment.
  • This elution fraction was equilibrated with a solution of 20 mM sodium phosphate / 150 mM NaCl (pH 7.0) in a volume 4 times the volume of the column, and a 150 cm / hr line was applied to a Q-Sepharose column (column volume; approximately 6.3 L). It was loaded at a flow rate and adsorbed. Subsequently, the column was washed with a solution of 20 mM sodium phosphate / 150 mM NaCl (pH 7.0) 4 times the column volume at the same flow rate, and then 20 mM sodium phosphate / 400 mM NaCl (pH 7.0) 5 times the column volume. ) The adsorbed protein was eluted with the solution.
  • the fraction eluted from the Q-Sepharose FF column was concentrated by ultrafiltration membrane concentration (exclusion limit molecular weight: 8,000). This was applied to a Superdex® 200 pg column (column volume; approximately 19 L, Amersham) equilibrated with 20 mM sodium phosphate / 150 mM NaCl (pH 6.0), gel filtered, and purified with recombinant human iduronic acid 2-sulfatase. Got the product.
  • peak A in FIG. 1 the main peak of human iduronic acid 2-sulfatase (peak A in FIG. 1).
  • peak 4 was considered to be Chinese hamster iduronic acid 2-sulfatase derived from CHO cells, and part of peak 6 was Chinese hamster N-acetylglucosamine.
  • 6 sulfatase, part of peak 7 was found to be Chinese hamster allylsulfatase A and Chinese hamster ⁇ -galactosidase A. Peaks 1-3 were not analyzed.
  • the purified product of human lysosomal enzyme is contaminated with proteins derived from mammalian cells that are different from humans, even in trace amounts, and it is considered that such purified product is administered to a patient by intravenous injection or the like. It is expected to cause an immune response and cause serious side effects and should be eliminated.
  • the structure of the lysosomal enzyme is conserved among mammalian cells and approximates its properties. Therefore, it was considered difficult to completely separate a human lysosomal enzyme from a heterogeneous lysosomal enzyme such as a Chinese hamster lysosomal enzyme.
  • a neomycin resistance gene cassette (about 2.2 kb) in which a neomycin resistance gene is arranged downstream of the SV40 promoter and an SV40 poly A signal sequence is further arranged downstream thereof is used to translate the endogenous iduronic acid 2-sulfatase gene in CHO cells.
  • Homologous recombination method using a DNA fragment of about 6.1 kb in total length inserted between the 5 ′ genomic sequence (about 1.4 kb) outside the region and the 3 ′ genomic sequence (about 2.5 kb) of the translation initiation codon Can disrupt the endogenous iduronic acid 2-sulfatase gene.
  • the gene-disrupted cells can be selected in a medium supplemented with neomycin, the selection is easy. Gene disruption can be confirmed by Northern blotting, and cells in which both iduronic acid 2-sulfatase genes present in homologous chromosomes are both disrupted can be selected.
  • a recombinant human lysosomal enzyme free of lysosomal enzyme contamination derived from an endogenous gene of the host cell, for example, recombinant human iduronic acid 2-sulfatase.

Abstract

La présente invention concerne un nouveau procédé de production d'enzymes lysosomales recombinantes, qui est apte à prévenir la contamination par des enzymes dérivées des gènes endogènes d'une cellule hôte. Ledit procédé est caractérisé en ce que des cellules, dans lesquelles des gènes codant pour des enzymes lysosomales endogènes ont été détruits, sont transformées par un vecteur d'expression qui incorpore des gènes codant pour les enzymes lysosomales humaines, et des enzymes lysosomales recombinantes sont produites au moyen desdites cellules transformées.
PCT/JP2011/054282 2010-03-01 2011-02-25 Procédé de production d'enzymes lysosomales recombinantes au moyen de cellules présentant une inactivation de gène WO2011108451A1 (fr)

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Cited By (7)

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WO2014017088A1 (fr) 2012-07-26 2014-01-30 Jcr Pharmaceuticals Co., Ltd. Procédé de production d'alpha-galactosidase a humaine recombinante
JP2015523072A (ja) * 2012-06-29 2015-08-13 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド 組換えイズロン酸２スルファターゼの精製
JP2015524249A (ja) * 2012-07-26 2015-08-24 Ｊｃｒファーマ株式会社 組換えヒトα−ガラクトシダーゼＡの製造方法
WO2016116966A1 (fr) * 2015-01-22 2016-07-28 Jcr Pharmaceuticals Co., Ltd. Procédé de purification d'une alpha-galactosidase a recombinante humaine à partir de matériel contenant des protéines contaminantes des cellules hôtes
WO2016193431A1 (fr) * 2015-06-05 2016-12-08 Laboratorios Del Dr. Esteve, S.A. Vecteurs dérivés de virus adéno-associés pour le traitement de mucopolysaccharidoses
JP2017014169A (ja) * 2015-07-03 2017-01-19 国立研究開発法人医薬基盤・健康・栄養研究所 ペプチド又はタンパク質の分画方法
US9719074B2 (en) 2012-06-29 2017-08-01 Shire Human Genetic Therapies, Inc. Cells for producing recombinant iduronate-2-sulfatase

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JP2015523072A (ja) * 2012-06-29 2015-08-13 シャイアー ヒューマン ジェネティック セラピーズ インコーポレイテッド 組換えイズロン酸２スルファターゼの精製
US11530393B2 (en) 2012-06-29 2022-12-20 Takeda Pharmaceutical Company Limited Compositions comprising iduronate-2-sulfatase
US9492511B2 (en) 2012-06-29 2016-11-15 Shire Human Genetic Therapies, Inc. Methods and compositions for treatment of hunter syndrome
US10344270B2 (en) 2012-06-29 2019-07-09 Shire Human Genetic Therapies, Inc. Methods and compositions for treatment of Hunter syndrome
US9719074B2 (en) 2012-06-29 2017-08-01 Shire Human Genetic Therapies, Inc. Cells for producing recombinant iduronate-2-sulfatase
US9617529B2 (en) 2012-07-26 2017-04-11 Jcr Pharmaceuticals Co., Ltd. Method for production of recombinant human alpha-galactosidase A
WO2014016873A1 (fr) * 2012-07-26 2014-01-30 Jcr Pharmaceuticals Co., Ltd. Procédé de production d'alpha-galactosidase a humaine recombinante
JP2015524249A (ja) * 2012-07-26 2015-08-24 Ｊｃｒファーマ株式会社 組換えヒトα−ガラクトシダーゼＡの製造方法
WO2014017088A1 (fr) 2012-07-26 2014-01-30 Jcr Pharmaceuticals Co., Ltd. Procédé de production d'alpha-galactosidase a humaine recombinante
WO2016116966A1 (fr) * 2015-01-22 2016-07-28 Jcr Pharmaceuticals Co., Ltd. Procédé de purification d'une alpha-galactosidase a recombinante humaine à partir de matériel contenant des protéines contaminantes des cellules hôtes
CN107208081A (zh) * 2015-01-22 2017-09-26 Jcr制药股份有限公司 用于从包含污染宿主细胞蛋白质的材料纯化重组人α‑半乳糖苷酶A的方法
US10385325B2 (en) 2015-01-22 2019-08-20 Jcr Pharmaceuticals Co., Ltd. Method for production of recombinant human alpha-galactosidase A from material containing contaminant host cell proteins by chromatography
CN107208081B (zh) * 2015-01-22 2020-07-14 Jcr制药股份有限公司 用于从包含污染宿主细胞蛋白质的材料纯化重组人α-半乳糖苷酶A的方法
WO2016117341A1 (fr) * 2015-01-22 2016-07-28 Jcr Pharmaceuticals Co., Ltd. Procédé de purification d'alpha-galactosidase a humaine de recombinaison à partir d'une matière contenant des protéines de cellules hôtes polluantes
WO2016193431A1 (fr) * 2015-06-05 2016-12-08 Laboratorios Del Dr. Esteve, S.A. Vecteurs dérivés de virus adéno-associés pour le traitement de mucopolysaccharidoses
US10617771B2 (en) 2015-06-05 2020-04-14 Universitat Autónoma De Barcelona Adenoassociated virus vectors for the treatment of mucopolysaccharidoses
RU2744593C2 (ru) * 2015-06-05 2021-03-11 Эстев Фармасьютикалс, С.А. Векторы на основе аденоассоциированных вирусов для лечения мукополисахаридозов
AU2016273343B2 (en) * 2015-06-05 2022-04-28 Esteve Pharmaceuticals, S.A. Adenoassociated virus vectors for the treatment of mucopolysaccharidoses
JP2017014169A (ja) * 2015-07-03 2017-01-19 国立研究開発法人医薬基盤・健康・栄養研究所 ペプチド又はタンパク質の分画方法

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