WO2006109695A1 - Composition comprenant une haptoglobine genetiquement modifiee - Google Patents

Composition comprenant une haptoglobine genetiquement modifiee Download PDF

Info

Publication number
WO2006109695A1
WO2006109695A1 PCT/JP2006/307374 JP2006307374W WO2006109695A1 WO 2006109695 A1 WO2006109695 A1 WO 2006109695A1 JP 2006307374 W JP2006307374 W JP 2006307374W WO 2006109695 A1 WO2006109695 A1 WO 2006109695A1
Authority
WO
WIPO (PCT)
Prior art keywords
haptoglobin
seq
amino acid
acid sequence
dna
Prior art date
Application number
PCT/JP2006/307374
Other languages
English (en)
Japanese (ja)
Inventor
Kazuya Yamano
Yutaka Kanda
Mitsuo Satoh
Original Assignee
Kyowa Hakko Kogyo Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kyowa Hakko Kogyo Co., Ltd. filed Critical Kyowa Hakko Kogyo Co., Ltd.
Publication of WO2006109695A1 publication Critical patent/WO2006109695A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a composition having a recombinant haptoglobin molecular force having an N-glycoside-linked complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is N-acetyl at the reducing end of the sugar chain.
  • the present invention relates to a haptoglobin composition which is a sugar chain and has its use, wherein fucose is bound to darcosamine.
  • Haptoglobin is a plasma glycoprotein with a molecular weight of 85,000 to 400,000 produced by mature granular leukocytes represented by eosinophils present in hepatocytes and lymph nodes. Usually, it exists in blood at a concentration of 0.2 to 3 g / L, and when hemoglobin is released into the blood by hemolysis, it specifically binds to form a complex (Non-patent Document 1). This complex is transported to the liver and then taken up into the cell by binding to receptors such as CD163 expressed in cells of the reticuloendothelial cell line, and to hepatoxylase by hemoxygenase. Metabolized (Non-Patent Documents 2 to 4).
  • the blood half-life of human haptoglobin is 3.5 to 5 days.
  • the half-life of the complex bound to hemoglobin is 10-30 minutes, and it is known that it is rapidly taken up and degraded. .
  • hemolysis occurs due to the occurrence of pathological conditions such as burns, burns, blood transfusions, open-heart surgery under extracorporeal circulation such as cardiopulmonary bypass, and hemoglobin in the blood exceeds haptoglobin, hemoglobin leaks from the glomeruli. This causes iron depletion from the body and oxidative vascular and renal tubule damage caused by free hemoglobin.
  • Haptoglobin preparations are used as therapeutic agents for such hemoglobinemia and hemoglobinuria.
  • haptoglobin has characteristics as an acute phase reaction protein. In infection, inflammation, tissue disruption, malignant tumor, etc., serum haptoglobin is markedly increased due to ectopic production other than liver such as eosinophils. It is known to be accepted (Non-patent Document 5).
  • Haptoglobin has a tetramer structure (hereinafter referred to as (a
  • This is a glycoprotein that can take a multimeric structure based on this tetrameric structure (FIG. 2) (Non-patent Document 6).
  • the alpha chain and the j8 chain of ptoglobin are single genes encoded in one open reading frame and exist in human chromosome 6 long arm 22.1 (Non-patent Documents 7 and 8).
  • the haptoglobin precursor which is the translation product of this single gene, is cleaved at one site in the cell that expresses haptoglobin, thereby generating an ⁇ chain and a j8 chain.
  • Hp 1 and Hp2 There are two types of human habutoglobin genes, Hp 1 and Hp2, and there are genetic polymorphisms (Fig. 1), so each human has three types of haptoglobin, Hpl-1, Hp2-1 and Hp2-2. One of them ( Figure 2). There is a racial difference in the genotype distribution of this haptoglobin.
  • Hp2-1 is 45-55%
  • Hp2-2 is 30-40%
  • Hpl-1 is 10-20%
  • Japanese is Hp2- It is known that 1 is 58%
  • Hp2-2 is 35%
  • Hpl-1 is 7%
  • about half of Africans are Hpl-1 (Non-Patent Documents 9 and 10).
  • the frequency is low, but the back mutation from the Hp2 gene to the Hpl gene due to intramolecular recombination occurs constantly in somatic cells, and it has been reported that it is in a chimeric state. (Non-patent document 11).
  • the human Hp2 gene is a gene produced by gene duplication of the exon 3 and 4 regions of the a-chain gene containing many cysteine residues of the Hpl gene (Fig. 1) (Non-patent Document 12). Therefore, ( ⁇ ) 2 having an Hp2 a chain can form a multimer via the free SH group of the cysteine residue generated by duplication (Fig. 2). This further increases the number of haptoglobin molecules. It is a cause of existence as a mixture of molecular species with molecular weights ranging from 85,000 to 400,000 in human plasma, and a wide range of physical and physical heterogeneity.
  • ( ⁇ ⁇ ) 2 having an Hpl a chain has a higher amount of hemoglobin binding per unit protein than ( ⁇ ⁇ ) 2 having an Hp2 a chain or a multimer (Non-patent Documents 13 and 10)
  • the haptoglobin-hemoglobin complex binds to the receptor expressed in reticuloendothelial cell line cells and is then taken up into the cell and metabolizes the bound hemoglobin. It has been reported that it is superior to ( ⁇ ) 2 having an a chain (Non-Patent Document 14).
  • haptoglobin can function as an antioxidant by suppressing the production of active oxygen radicals via iron in hemoglobin by binding to free hemoglobin.
  • Non-patent Document 10 The function as an antioxidant is also Hp2 a chain.
  • a j8) 2 having an Hpl a chain is higher than ( ⁇ ) 2 having a phenotype and multimers.
  • Non-patent Document 10 it has been reported that in humans of the Hp2-2 type gene polymorphism, vitamin C, which has a high abundance of iron ions in serum, has poor stability (Non-patent Documents 15 and 16).
  • haptoglobulin having an Hp2 a chain is sufficient to transfer to yarn and tissue. I can not expect. Therefore, in order to provide a uniform bioactive haptoglobin preparation, it is desired to adjust the amount of hemoglobin binding per unit preparation protein and make the molecular species composition constant.
  • Non-patent Documents 17 to 20 It has been reported that the sugar chain of haptoglobin has a low fucose modification rate of a few percent or less in healthy individuals, but it increases with canceration (Non-patent Documents). 21, 22).
  • haptoglobin preparations are produced from the plasma of many thousands of healthy individuals. For this reason, these preparations may cause transmission of infections such as various hepatitis viruses, AIDS viruses, human parvoviruses, and prions that cause mutant Creutzfeldt's Jacob disease. This cannot be completely denied. In addition, it is difficult to completely overcome the physical and physiological non-uniformity because it is necessary to use plasma that can be obtained from unspecified different healthy human power as a raw material. . From this point of view, it is instructed that the use of haptoglobin preparations should be kept to the minimum necessary after careful examination of the therapeutic need. The name and address, the date of administration, and the serial number of the administered product must be recorded and stored for at least 20 years.
  • Patent Document 1 Non-patent Document 1
  • Patent Documents 23 and 24 the sugar chain structure bound to haptoglobin produced by these host cells is significantly different from the sugar chain structure of human plasma-derived haptoglobin, and has the same activity as human plasma-derived haptoglobin.
  • haptoglobins having different sugar chain structures are not preferable as pharmaceuticals in terms of antigenicity, blood dynamics, and stability.
  • Non-patent Document 25 CHO cells derived from Chinese hamster ovary that have already been used in pharmaceutical manufacturing and have been proven to have no sugar chain structure that causes problems when administered to humans are used as host cells to produce recombinant haptoglobin.
  • Non-patent Document 25 An example has been reported (Non-patent Document 25). However, it is bound to haptoglobin obtained by the production method and fucose is bound to most of the sugar chains, and this structure is similar to the abnormal haptoglobin observed in the plasma of cancer-bearing patients. It is known that The causal relationship between fucose modification to sugar chains of haptoglobin and the carcinogenicity is still unknown.
  • haptoglobin In the treatment of patients, the single dose of haptoglobin preparation is about 5.7 g, so haptoglobin In order to administer to patients, it must be serious. In addition, because of the high physiological activity of hemoglobin, buttoglobin, not only is it related to canceration, but also considers the effect on the biodistribution of haptoglobin itself and its complex with hemoglobin due to a significant change in sugar chain structure. It is left as an issue to be done. Therefore, there is a need for a recombinant haptoglobin that has a pharmacological effect equivalent to or higher than that of human plasma-derived haptoglobin, and that can reduce the risk of virus infection, which is a drawback of current blood products, and heterogeneity. It is. Patent Document 1: Japanese Patent Publication No. 46068 2001
  • Non-Patent Document 2 Proc. Natl. Acad. Sci. USA, 61, 748 (1968)
  • Non-Patent Document 3 J. Biol. Chem., 255, 9616 (1980)
  • Non-Patent Document 4 Circ. Res., 92, 1193 (2003)
  • Non-Patent Document 5 Clin. Lab., 46, 547 (2000)
  • Non-Patent Document 6 Redox Rep., 6, 379 (2001)
  • Non-Patent Document 7 Hum. Genet., 42, 61 (1978)
  • Non-Patent Document 8 Proc. Natl. Acad. Sci. USA, 80, 5875 (1983)
  • Non-Patent Document 9 The Second Series of Pharmaceutical Research and Development 'Bio od Product "20, 215 (1992)
  • Non-Patent Document 10 Clin. Lab., 46, 547 (2000)
  • Non-Patent Document 12 Redox Report, 6, 119 (2001)
  • Non-Patent Document 13 Vox Sang, 10, 320 (1965)
  • Non-Patent Document 14 Circ. Res., 92, 1193 (2003)
  • Non-Patent Document 15 Am. J. Clin. Nutr., 66, 606 (1997)
  • Non-Patent Document 16 AIDS 12, 1027 (1998)
  • Non-Patent Document 17 J. Biol. Chem., 258, 7858 (1983)
  • Non-Patent Document 18 Clin. Chim. Acta, 227, 69 (1994)
  • Non-Patent Document 19 Biochim. Biophys. Acta, 953, 345 (1988)
  • Non-Patent Document 20 Glycoconjugates. Proc. 6th Int. Sym. Glyco. Tokyo, 275 (1981)
  • Non-Patent Document 21 Cancer Letters, 66, 43 (1992)
  • Non-Patent Document 22 The Second Series of Pharmaceutical Research and Development 'BL oodProduct "20, 215 (1992)
  • Non-Patent Document 23 DNA, 5, 129 (1986)
  • Non-Patent Document 24 Mol. Biol. Rep., 13, 225 (1989)
  • Non-Patent Document 25 Proteomics, 4, 2221 (2004) Disclosure of the invention
  • the present invention relates to the following (1) to (24).
  • a composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain binds N-acetyldarcosamine at the reducing end of the sugar chain.
  • a haptoglobin composition which is a sugar chain.
  • the N-glycoside-linked complex type sugar chain is a sugar chain in which N-acetylyldarcosamine at the reducing end of the sugar chain is a sugar chain in which position 1 of fucose is not ⁇ -bonded.
  • Haptoglobin composition is a sugar chain in which N-acetylyldarcosamine at the reducing end of the sugar chain is a sugar chain in which position 1 of fucose is not ⁇ -bonded.
  • Haptoglobin force A protein consisting of the following (a) to (i) group forces that also have forces, and the following (j) to (l) polypeptides that also have group forces that have selected forces.
  • Haptoglobin force The following (a), (b), (c), (d), (e) and (1) Group force of force The above-mentioned (1) or ( The haptoglobin composition described in 2).
  • the host cell is the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or the N-glycidyl-linked N-acetylyldarcosamine at the 6-position of the reducing glycan is located at position 1 of fucose ⁇
  • the transformant according to (5) above, which is a cell whose genome has been altered so that the activity of the enzyme involved in the modification of the sugar chain to be bound is lost.
  • the host cell is the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose, or the first position of fucose at position 6 of ⁇ ⁇ -acetylcylcosamine at the reducing end of ⁇ -glycosidic complex glycan
  • the transformant according to (6) above, wherein the allele on the genome of the enzyme involved in the sugar chain modification to be bound is a knocked-out cell.
  • amino acid sequence represented by SEQ ID NO: 8 one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have GDP-mannose 4,6-dehydratase activity protein;
  • GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein in which the following (a), (b) and (c) group forces are selected (8) ).
  • amino acid sequence represented by SEQ ID NO: 10 one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence power is GDP-4-keto-6-deoxy-D- A protein having mannose-3,5-epimerase activity;
  • (c) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity protein.
  • the above-mentioned (8) GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is a protein encoded by DNA selected from the following (a) and (b) group forces that also include forces (8) ).
  • N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end is an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to a 1,6-fucosyltransferase
  • a 1,6-fucosyltransferase is a protein for which a group force consisting of the following (a), (b), (c), (d), (e) and (1) is also selected (13 ).
  • amino acid sequence represented by SEQ ID NO: 14 one or more amino acids are deleted, substituted, inserted, and have Z or added amino acid sequence ability, and have an ⁇ 1,6-fucosyltransferase activity.
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and having a 1,6-fucosyltransferase activity.
  • a 1, 6-fucosyltransferase is a protein encoded by DNA selected from the group consisting of the following (a), (b), (c) and (d) force: Transformant.
  • Lentil lectin LCA Lientil Agglutinin from Lens Culinaris
  • Herochawantake lectin AAL Lectin from Aleuria aurantia
  • the host cell has the following (a), (b), (c), (d), (e), (£), (g), (h), (i) and (j) forces The transformant according to any one of (5) to (18) above, which is a cell selected from a group.
  • the transformant according to any one of (5) to (19) above is cultured in a medium, a haptoglobin composition is produced and accumulated in the culture, and the haptoglobin composition is collected from the culture
  • the manufacturing method of a haptoglobin composition including a process.
  • a medicament comprising the haptoglobin composition according to any one of (1) to (4) and (21) as an active ingredient.
  • a therapeutic agent for hemoglobinemia and hemoglobinuria comprising the haptoglobin composition according to any one of (1) to (4) and (21) as an active ingredient.
  • a disease associated with acid-induced vascular injury due to blood free hemoglobin comprising as an active ingredient the haptoglobin composition according to item 1 of (1) to (4) and (21) above Therapeutic agent.
  • composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is N- A haptoglobin composition which is a sugar chain in which fucose is bound to acetylyldarcosamine is provided.
  • FIG. 1 shows the structure of HP-1 type and HP-2 type haptoglobin.
  • FIG. 2 shows the structure of the haptoglobin complex in HP1-1 type, HP2-1 type, and HP2-2 type.
  • FIG. 3 shows a method for preparing plasmid pKAN-HP1.
  • FIG. 4 shows a method for preparing plasmid pKAN-HP2.
  • the composition of the present invention also comprises a recombinant haptoglobin molecule having an N-glycoside-bonded complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is N-acetyl at the reducing end of the sugar chain.
  • a haptoglobin composition (hereinafter referred to as “the composition of the present invention”), which is a sugar chain in which fucose is bound to darcosamine, is an N-glycoside-linked complex sugar chain reducing end N-a. Any composition is included as long as it is a haptoglobin composition in which fucose is bound to cetyldarcosamine! /, Na! / And also has a recombinant haptoglobin molecular force.
  • haptoglobin includes any glycoprotein having affinity for hemoglobin and a haptoglobin receptor and having hemoglobin metabolic activity.
  • haptoglobin examples include a polypeptide selected from the group consisting of (a) to (i) below the haptoglobin power and a polypeptide selected from the group consisting of (j) to (l) below: Examples include proteins.
  • polypeptide having an amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4 (j) a polypeptide having an amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4; (k) one or more amino acids deleted in the amino acid sequence represented by positions 103 to 347 of SEQ ID NO: 4, A polypeptide having a substitution, insertion and Z or added amino acid sequence, and having hemoglobin metabolic activity in combination with the above-mentioned (a) to (polypeptides having a group strength of 0);
  • DNA encoding the amino acid sequence of haptoglobin is stringent with DNA having the base sequence represented by SEQ ID NO: 1, 2 or 3, and DNA having the base sequence represented by SEQ ID NO: 1, 2 or 3.
  • Examples include DNA that encodes a protein that undergoes nobbreviation and hybridization under certain conditions and has hemoglobin metabolic activity.
  • the DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, or 3, or a fragment thereof.
  • Koguchi-1 'hybridization method plaque' hybridization method is "means DNA obtained by using Southern blot hybridization method, etc., specifically, colony or Hybridization was performed at 65 ° C in the presence of 0.7 to 1.
  • OM sodium chloride using a filter on which plaque-derived DNA was immobilized, and then an SSC solution (1 to 2 times in concentration) was added.
  • the concentration of SSC solution is 150mM sodium chloride and 15mM sodium citrate), and DNA can be identified by washing the filter under 65 ° C conditions. .
  • DNA capable of hybridizing is DNA having at least 60% or more homology with the nucleotide sequence represented by SEQ ID NO: 1, 2, or 3, preferably 70% or more, more preferably 80% or more, and still more preferably May be DNA having a homology of 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • amino acids in the amino acid sequence represented by SEQ ID NO: 4, 5, or 6, one or more amino acids
  • a protein that has amino acid sequence ability with deletion, substitution, insertion and Z or addition of mino acid, and also has a hemoglobin metabolic activity is the molecular 'Clooung 2nd edition, current' Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34,315 (1985), Nucleic Acids Research, 13 , 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc., for example, represented by SEQ ID NO: 4, 5, or 6 It means a protein that can be obtained by introducing a site-specific mutation into DNA encoding a protein having an amino acid sequence.
  • the number of amino acids to be deleted, substituted, inserted, and Z or added is 1 or more, and the number is not particularly limited. For example, it is 1 to several tens, preferably 1 to 20, more preferably 1 to: L0, and further preferably 1 to 5.
  • the protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4, 5, or 6 and having hemoglobin metabolic activity is BLAST [J. Mol. Biol, 215, 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)], etc., and the amino acid sequence described in SEQ ID NO: 4, 5 or 6 At least 80% or more, preferably 85% or more, more preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more. Means that.
  • Hemoglobin metabolic activity refers to the binding of haptoglobin force to free hemoglobin to form a complex, and the complex is expressed as a receptor for the hemoglobin-habu globin complex expressed in the reticuloendothelial cell line of the liver. It binds and is taken up into cells after binding, and has the activity of metabolizing heme generated by the degradation of hemoglobin into pyrilbin.
  • N-glycoside-linked sugar chains bound to glycoproteins are known to have a common core structure represented by the following structural formula (I) in any of the various structures. .
  • the end of the sugar chain that binds to asparagine is the reducing end, and the opposite side is non- It is called the reducing end.
  • the N-glycoside-linked sugar chain is a high mannose type in which only mannose binds to the non-reducing end of the core structure, and galactose —N-acetyldarcosamine (hereinafter referred to as Ga ⁇ GlcNAc) 1) in parallel, and more than one branch, and a complex type having a structure such as sialic acid, Neusecting N-acetylethylcosamine on the non-reducing end side of Ga ⁇ GlcNAc, It is known that there are hybrid types having both a hymannose type and a complex type branch on the non-reducing end side of the core structure.
  • the j8 chain of the haptoglobin molecule constituting the composition of the present invention has four additional sequences of N-glycoside-linked sugar chains, and N-glycoside-linked sugar chains are bound to these sites.
  • Specific examples of the N-glycoside-bonded sugar chain that binds to habtoglobin include the aforementioned N-glycoside-bonded complex sugar chains.
  • the N-glycoside-bonded complex sugar chain that binds to the haptoglobin molecule includes a large number of sugar chains including the core structure represented by the structural formula (I), and therefore there are many combinations of sugar chains. It will be.
  • the composition of the present invention is a composition having a genetically modified haptoglobin molecular force having an N-glycoside-bonded complex sugar chain, wherein the N-glycoside-bonded complex sugar chain is the sugar chain reducing end.
  • a haptoglobin molecular force having a single sugar chain structure or a haptoglobin having a plurality of different sugar chain structures may be used. Consists of molecules.
  • N-glycoside-linked complex type sugar chain is a sugar chain in which fucose is not bound to N-acetylyldarcosamine at the reducing end. Specifically, position 1 of fucose is an N-glycoside-linked complex type sugar. The 6-position of the N-acetylyldarcosamine in the chain (X-bonded!
  • fucose is bonded to ⁇ ⁇ -acetyldarcosamine at the sugar chain reducing end.
  • An unacceptable sugar chain means that fucose is not substantially bound to the sugar chain, and preferably means that the fucose content is 0%.
  • the fact that fucose is not substantially bound means that fucose cannot be substantially detected in the sugar chain analysis described in 4 below. “Substantially undetectable” means below the detection limit of measurement.
  • the composition of the present invention has a long half-life in blood when administered in vivo compared to haptoglobin in which fucose is bound to N-glycidyl darcosamine at the N-glycoside-linked complex type sugar chain reducing end. .
  • the transformant of the present invention includes any transformant as long as it is capable of producing the composition of the present invention.
  • a specific example is a transformant obtained by introducing a DNA encoding a haptoglobin molecule into a host cell such as (a) or (b) below.
  • Enzymes involved in the synthesis of intracellular sugar nucleotides GDP-fucose include: GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose-3,5 -Epimerase (FX).
  • the GDP-mannose 4,6-dehydratase includes a protein encoded by the following DNA (a) or (b), or a protein (c), (d) or (e) below. .
  • amino acid sequence represented by SEQ ID NO: 8 one or more amino acids are deleted, substituted, or inserted A protein having amino acid sequence ability with added and Z or added, and having GDP-mannose 4,6-dehydratase activity;
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, and having GDP-mannose 4,6-dehydratase activity.
  • GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase is:
  • Examples include the protein encoded by DNA (a) or (b) below, or the protein (c), (d) or (e) below.
  • amino acid sequence represented by SEQ ID NO: 10 one or more amino acids are deleted, substituted, inserted and Z or added, and the amino acid sequence power is GDP-4-keto-6-deoxy-D-mannose.
  • (e) It consists of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and has GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity Protein.
  • N-glycoside-linked complex type sugar chain N-acetylyldarcosamine at the reducing end has an enzyme involved in sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of a 1,6-fucosyltransferase For example.
  • ⁇ 1,6-fucosyltransferase is a protein encoded by the following DNA (a), (b), (c) or (d), or (, (£), (g), (H), (0 or (j) protein, etc.).
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 14, and having a 1,6-fucosyltransferase activity.
  • DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having a nucleotide sequence represented by SEQ ID NO: 7, 9, 11, or 12, or a fragment thereof.
  • DNA obtained by using the Koguchi-ichi 'hybridization method, plaque' hybridization method, Southern hybridization method, etc. as a probe, specifically derived from colonies or plaques.
  • OM sodium chloride 0.1 to 2 times the concentration of SSC solution (The composition of the SSC solution with a 1-fold concentration consists of 150 mM sodium chloride and 15 mM sodium quenate), and DNA can be identified by washing the filter under 65 ° C conditions.
  • DNA that can be hybridized under stringent conditions is specifically DNA having at least 60% homology with the nucleotide sequence represented by SEQ ID NO: 7, 9, 11 or 12, preferably 70% or more More preferred is DNA having a homology of 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more.
  • the amino acid sequence represented by SEQ ID NO: 8 consists of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and Z or added, and the GDP-mannose 4,6-dehydratase activity
  • GDP-4-keto-6-deoxy-D -A protein having mannose-3,5-epimerase activity or an amino acid sequence having one or more amino acids deleted, substituted, inserted and Z or added in the amino acid sequence represented by SEQ ID NO: 13 or 14.
  • the number of amino acids to be deleted, substituted, inserted and Z or added is 1 or more and the number is not particularly limited, but the above-mentioned site-specific mutagenesis method, etc.
  • the number is such that it can be deleted, substituted or added by, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5. is there.
  • the present invention comprises an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8, 10, 13 or 14, and has GDP-mannose 4,6-dehydratase activity. , GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase activity or ⁇ 1,
  • the amino acid sequence represented by SEQ ID NO: 8, 10, 13 or 14 and BLAST [J. Mol.
  • a host cell lacking the above-mentioned enzyme activity that is, an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylyldarcosamine at the N-glycoside-linked complex sugar chain reducing end
  • an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-acetylyldarcosamine at the N-glycoside-linked complex sugar chain reducing end By introducing DNA encoding a haptoglobin molecule into a host cell whose genome has been altered so that the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is linked to a at position 6 is a It is possible to obtain a transformant producing the composition.
  • the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose, or N-acetylcolcamine at the reducing end of the N-daricoside-linked complex sugar chain, position 6 of fucose is ⁇ -linked
  • a mutation is introduced into the expression regulatory region of the gene so as to eliminate the expression of the enzyme, or the function of the enzyme It means that a mutation is introduced into the amino acid sequence of the gene so as to disappear.
  • Introducing mutation means that the base sequence on the genome is deleted, substituted, inserted, and deleted or added, and the base sequence is modified, completely suppressing the expression or function of the modified genomic gene.
  • Knock out to do it is one in which all or part of the target gene has been deleted from the genome. It can be knocked out by removing the genomic region of the etason containing the start codon of the target gene.
  • any method can be used as long as the target genome can be modified.
  • any lectin that can recognize the sugar chain structure can be used. Specific examples of this are: Lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), Endumame lectin PS A (Peum sativum-derived PeaLectin), Broad bean lectin VFA (Agglutini n from Vicia faba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
  • a cell resistant to lectin refers to a cell whose growth is not inhibited even when an effective concentration of lectin is given.
  • the effective concentration is not less than the concentration at which cells before the genomic gene is modified (hereinafter also referred to as “parent cell”) cannot grow normally, preferably the concentration at which cells before the modified genomic gene cannot grow , More preferably 2 to 5 times, still more preferably 10 times, and most preferably 20 times or more.
  • the effective concentration of lectin whose growth is not inhibited may be appropriately determined depending on the cell line, but is usually 10 / zg / ml to 10mg / ml, preferably 0.5mg / ml to 2.0mg. / ml.
  • any cell that can express the composition of the present invention can be used.
  • yeast, animal cells, insect cells, plant cells and the like can be mentioned. Specific examples include those described in 3. below.
  • animal cells include CHO cells derived from Chinese omster ovary tissue, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0- Examples include Agl4 cells, Syrian hamster kidney tissue-derived BHK cells, human leukemia cell lines Namalba cells, embryonic stem cells, and fertilized egg cells.
  • a host cell used for producing a recombinant glycoprotein pharmaceutical examples include embryonic stem cells or fertilized egg cells used to produce non-human transgenic animals that produce pesticides, and plant cells used to produce transgenic plants that produce recombinant glycoprotein drugs. It is done.
  • an enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose, or N-glycyl-linked complex N-acetylyldarcosamine at the 6-position of N-acetylyldarcosamine It includes cells prior to the application of a technique for altering the genomic gene of an enzyme involved in sugar chain modification in which position 1 is OC-linked. For example, the following cells are preferable.
  • NS0 cell parent cell lines are described in the literature such as Bio / Technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology No. 1 Bioengineering (Biotechnol. Bioeng.), 73, 261, (2 001), etc. NS0 cells are listed.
  • NS0 cell line (RCB0213) registered with the RIKEN Cell Development Bank, or sub-strains obtained by acclimatizing these strains to various serum-free media are also included.
  • CHO-K1 strain ATCC CCL-61
  • DUXB11 strain ATCC CRL-9096
  • Pro-5 strain ATCC CRL-1781 registered in ATCC
  • commercially available CHO-S strain (Lifetechnologi es Cat # l 1619), or sub-strains made by adapting these strains to various serum-free media can give.
  • the parent cell of rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell includes a cell line established from Y3 / Ag 1.2.3 cell (ATCC CRL-1631). Specific examples are YB2 / 3HL.P2.G11.16Ag. Described in documents such as J. Cell. Biol., 93, 576 (1982), Methods Enzymol. 73B, 1 (1981). There are 20 cells. In addition, YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL-1662) registered in ATCC or sub-strains obtained by acclimating these strains to various serum-free media are also included.
  • a gene encoding a haptoglobin Hpl gene was introduced into a CHO cell into which a gene encoding ⁇ 1,6-fucosyltransferase was knocked out.
  • examples thereof include the HP1KO strain, which is a transformant, and the HP2KO strain, which is a transformant in which a gene encoding haptoglobin Hp2 is introduced into a CHO cell into which a gene encoding a 1,6-fucosyltransferase has been knocked out.
  • HP1KO and HP2KO strains were established on February 17, 2005 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Tsukuba Rinto, Tsukuba 1-chome, 1-Chuo 6th, Ibaraki, Japan). Deposited as FERM BP-10249 and FERM BP-10250, respectively.
  • the transformant of the present invention has a hemoglobin binding activity equivalent to that of the haptoglobin composition obtained from the parent cell line, and has a longer blood half-life than the haptoglobin composition obtained from the parent cell line.
  • a buttoglobin composition can be produced.
  • the binding activity of the complex to the receptor present in reticuloendothelial cells and the half-life in blood were determined by measuring the binding activity of the known hemoglobin and haptoglobin receptor in vitro tests, model animals such as mice and rats. It can be measured using an in vivo test used or a clinical test using humans (Clin. Chem., 42, 1589 (1996), Eur. J. Clin. Chem. Biochem., 35, 647 ( 1997), Nature, 409, 198 (2001), J. Biol. Chem., 279, 51561 (2004), Modern Medicine, 30, 656 (1975), Modern Medicine, 30,879 (1975), Res. Exp.
  • the host cell used for producing the haptoglobin thread and the composition of the present invention can be produced by the method described below.
  • the host cell used for the preparation of the haptoglobin composition of the present invention is an enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or N-glycoside-linked complex sugar chain-reducing terminal N-acetylyldarcosamine 6
  • enzyme related to fucose modification By using the gene disruption method targeting the gene of the enzyme involved in the sugar chain modification in which position 1 of fucose binds to the position (hereinafter referred to as “enzyme related to fucose modification”) Can do.
  • GDP-fucose examples include GDP-mannose 4,6-dehydratase (hereinafter referred to as “GMD”), GDP-4-keto-6-deoxy -D-mannose-3,5-epimerase (hereinafter referred to as “Fx”).
  • GMD GDP-mannose 4,6-dehydratase
  • Fx GDP-4-keto-6-deoxy -D-mannose-3,5-epimerase
  • enzymes involved in glycosylation in which the 1-position of fucose is a-linked to the 6-position of N-glycidyl darcosamine at the N-glycoside-bonded glycan reducing end examples include ⁇ 1,6-fucosyl Examples include transferase and a-L-fucosidase.
  • the gene herein includes DNA or RNA.
  • any method can be used as the gene disruption method as long as it can destroy the gene of the target enzyme.
  • Examples include the antisense method, ribozyme method, homologous recombination method, RNA-DNA oligonucleotide method (hereinafter referred to as “RDO method”), RNA interference method (hereinafter referred to as “RNAi method”). ), A method using a retrovirus, a method using a transposon, and the like. These will be specifically described below.
  • the host cell used to produce the haptoglobin composition of the present invention targets an enzyme gene related to fucose modification, and is described in Cell Engineering, 12, 239 (1993), Bio-Z Technology (BIO / TECHNOLOGY), 17, 1097 ( 1999), Human 'Molecular ⁇ ⁇ ⁇ Genetics (Hum.Mol. Genet.), 5, 1083 (1995), Cell engineering, 13, 255 (1994), Proceedings' o B. The National. Academia. Ob. Science ( proc . Natl. Acad. Sci. USA), 96, 1
  • cDNA or genomic DNA encoding an enzyme related to fucose modification is prepared.
  • an antisense gene or ribozyme construct of appropriate length including the DNA part encoding the enzyme related to fucose modification, the part of the untranslated region or the intron part.
  • D prepared in order to express the antisense gene or ribozyme in cells
  • a recombinant vector is prepared by inserting the NA fragment or full length downstream of the promoter of an appropriate expression vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • a host cell used for preparing the haptoglobin composition of the present invention By selecting a transformant using the activity of an enzyme related to fucose modification as an indicator, a host cell used for preparing the haptoglobin composition of the present invention can be obtained. It is also possible to obtain a host cell used for producing the haptoglobin composition of the present invention by selecting a transformant using the sugar chain structure of a glycoprotein on the cell membrane or the sugar chain structure of a produced glycoprotein molecule as an index. it can.
  • the host cell used for preparing the haptoglobin composition of the present invention has a gene for an enzyme related to target fucose modification, such as an enzyme, an animal cell, an insect cell, or a plant cell. Any material can be used. Specific examples include host cells described in 3 below.
  • the expression vector is capable of autonomous replication in the above host cell, or can be integrated into the chromosome and contains a designed antisense gene or a promoter at a position where a ribozyme can be transcribed. .
  • the expression vector described in 3 below can be mentioned.
  • the method for introducing a gene into various host cells the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
  • Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the following methods.
  • Examples of a method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • Examples of a method for preparing cDNA encoding an enzyme related to fucose modification include the methods described below.
  • Total RNA or mRNA is prepared from the tissues or cell strength of various host cells.
  • a cDNA library is prepared from the prepared total RNA or mRNA.
  • a degenerative primer is prepared, and a gene fragment encoding the enzyme related to fucose modification is obtained by PCR using the prepared cDNA library as a saddle type To do.
  • a cDNA library can be screened to obtain DNA encoding an enzyme related to fucose modification.
  • V or human or non-human animal tissue or cell force may be prepared as follows.
  • thiocyanic acid As a method for preparing total RNA of human or non-human animal tissues or cells, thiocyanic acid can be used.
  • examples of a method for preparing mRNA as total RNA poly (A) + RNA include an oligo (dT) -fixed cellulose column method (Molecular 'Cloung 2nd edition).
  • mRNA can be prepared by using a commercially available kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
  • kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
  • a cDNA library is prepared from the prepared human or non-human animal tissue or cell mRNA.
  • the cDNA library can be prepared by the method described in Molecular 'Crowing 2nd Edition, Current Protocols in Molecular Biology, A Laboratory Manual, 2nd Ed. (1989), etc., or a commercially available kit. For example, a method using Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies Neeri, ZAP—cDNA bynt hesis Kit (STRATAGENE)) can be mentioned.
  • Escherichia coli K12 strain is used as a cloning vector for preparing a cDNA library.
  • Any phage vector or plasmid vector can be used as long as it can replicate autonomously.
  • ZAP Express [STRATAGENE, Strategies,, 58
  • Escherichia coli is preferably used. Specifically, Escherichia coli XLl-Blue MRF '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 "Genetics, 39, 440 (1954) 1, Escherichia coli Y108 8 [ Science, 222, 778 (1983) 1, Escherichia coli Yl 090 "Science, 222,778 (1983) 1, Escherichia coli NM522" Journal, Ob, Molecura ⁇ Bio Mouth 0.
  • the obtained gene fragment is DNA encoding an enzyme related to fucose modification.
  • an enzyme related to fucose modification for example, Sanger et al.'S dideoxy method [Proceedings of the National] ⁇ Academ ⁇ ⁇ ⁇ Ob ⁇ Science (Pro Natl. Ac ad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems) etc.
  • Sanger et al.'S dideoxy method [Proceedings of the National] ⁇ Academ ⁇ ⁇ ⁇ Ob ⁇ Science (Pro Natl. Ac ad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems) etc.
  • a colony hybridization or a plaque hybridization is synthesized from cDNA or cDNA library synthesized from mRNA contained in tissues or cells of human or non-human animals. -Nu 2nd edition) etc. can be used to obtain DNA for enzymes related to fucose modification.
  • a cDNA or cDNA library synthesized from mRNA contained in human or non-human animal tissues or cells using the primers used to obtain a gene fragment encoding an enzyme related to fucose modification as a saddle type. Amplify using the PCR method to repair fucose
  • the base sequence of the DNA encoding the enzyme related to the obtained fucose modification can be determined by a commonly used base sequence analysis method such as Sanger et al.'S dideoxy method [procedings ⁇ of ⁇ the 'national ⁇ academia ⁇ By analyzing using a base sequence analyzer such as ⁇ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems), The base sequence of the DNA can be determined.
  • a base sequence analyzer such as ⁇ Ob Science (Pro Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA Sequencer (Applied Biosystems).
  • a homology search program such as BLAST is used to search a base sequence database such as Genbank, EMBL, and DDBJ. It can also be confirmed that the gene encodes an enzyme related to fucose modification.
  • Examples of the base sequence of the gene encoding the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the base sequence set forth in SEQ ID NO: 7 or 9.
  • N-glycoside-linked complex-type sugar chain-reducing terminal N-acetylcylcosamine obtained by the above method encodes an enzyme that encodes an enzyme involved in sugar chain modification in which position 1 of fucose is oc-bonded to position 6.
  • Examples of the base sequence include the base sequence described in SEQ ID NO: 11 or 12.
  • DNA synthesis using the phosphoramidite method is performed.
  • a DNA synthesizer such as model machine 392 (manufactured by Perkin Elmer)
  • cDNA of an enzyme related to fucose modification can also be obtained.
  • Examples of a method for preparing genomic DNA of an enzyme related to fucose modification include the methods described below.
  • genomic DNA of an enzyme related to fucose modification can be obtained by using a genomic DNA library screening system (Genome Systems), Unigen GenomeWalker TM Kits (CLONTECH), or the like.
  • the obtained DNA base sequence encoding the enzyme related to fucose modification is usually used.
  • the base sequence of the DNA can be determined.
  • a homology search program such as BLAST is used to search base sequence databases such as Genbank, EMBL and DDBJ. It is also possible to confirm that the gene encodes an enzyme related to fucose modification.
  • nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose obtained by the above method include the nucleotide sequences set forth in SEQ ID NOs: 15, 16, 17, and 18.
  • Examples thereof include the base sequence set forth in SEQ ID NO: 19.
  • the haptoglobin composition of the present invention is prepared by directly introducing into the host cell an antisense oligonucleotide or ribozyme designed based on the base sequence of the enzyme related to fucose modification without using an expression vector. The host cell used to do this is obtained.
  • Antisense oligonucleotides or ribozymes can be prepared by conventional methods or DNA synthesizers. Specifically, it corresponds to a continuous 5 to 150 bases, preferably 5 to 60 bases, more preferably 10 to 40 bases in the base sequences of cDNA and genomic DNA encoding the enzyme related to fucose modification. Based on the sequence information of the oligonucleotide having the sequence, an oligonucleotide (antisense oligonucleotide) corresponding to a sequence complementary to the oligonucleotide or a ribozyme containing the sequence of the oligonucleotide can be synthesized and prepared.
  • oligonucleotide derivatives examples include oligo RNA and derivatives of the oligonucleotide (hereinafter referred to as oligonucleotide derivatives).
  • Oligonucleotide derivatives include oligonucleotide derivatives in which phosphodiester bonds in oligonucleotides are converted to phosphorothioate bonds, and phosphodiester bonds in oligonucleotides are converted to ⁇ 3'- ⁇ 5 'phosphoramidate bonds.
  • Oligonucleotide derivatives oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which the uracil in the oligonucleotide is replaced with C-5 propylene uracil, in the oligonucleotide Derivative uracil in which uracil is substituted with C-5 thiazoleuracil, cytosine in the oligonucleotide is substituted with C-5 propylcytosine, and the derivative is cytosine in the oligonucleotide.
  • the host cell used for preparing the haptoglobin composition of the present invention can be prepared by targeting a gene of an enzyme related to fucose modification and modifying the target gene on the chromosome using a homologous recombination method. .
  • Genomic DNA of an enzyme related to fucose modification is prepared.
  • a target vector for homologous recombination of the target gene to be modified (for example, a structural gene of an enzyme related to fucose modification or a promoter gene) is prepared.
  • a host cell used for preparing the haptoglobin composition of the present invention by introducing the prepared target vector into a host cell and selecting a cell that has undergone homologous recombination between the target gene on the chromosome and the target vector. Can be produced.
  • any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • Examples thereof include a method for preparing genomic DNA described in (a).
  • nucleotide sequence of the genomic DNA of the enzyme involved in the synthesis of the intracellular sugar nucleotide GDP-fucose obtained by the above method for example, the salts described in SEQ ID NOs: 15, 16, 17 and 18 Examples of the base sequence.
  • target vector for homologous exchange of target genes on chromosomes, Gene Targ eting, A Practical Approach, IRLPress at Oxford University Press (1993), noisyo-Muual Series 8 Gene Targeting, ES cells were used. Production of mutant mice (Yodosha) (1995) and the like.
  • the target vector can be either a replacement type or an insertion type.
  • the method for introducing a recombinant vector suitable for various host cells described in 3 below can be used.
  • Examples of methods for efficiently selecting homologous recombinants include Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Bio-European Series 8 Gene Targeting, Production of mutant mice using ES cells Methods such as positive selection, promoter selection, negative selection, poly A selection described in (Yodosha) (1995) and the like can be used. Methods for selecting the desired homologous recombinants from the selected cell lines include the Southern hybridization method (Molequila's Cloning 2nd edition) for genomic DNA and the PCR method [PCR] 'Protocols (PCR Protocols), Academic Press (1990)].
  • the host cell used for preparing the haptoglobin composition of the present invention can be prepared as follows, for example, by targeting the gene of an enzyme related to fucose modification and using the RDO method.
  • cDNA or genomic DNA of an enzyme related to fucose modification is prepared using the method described in 1 (1) (a) above.
  • the portion encoding the enzyme related to fucose modification Based on the determined DNA sequence, the portion encoding the enzyme related to fucose modification, non- Design and synthesize an RDO construct of appropriate length including the translation region or intron.
  • the synthesized RDO is introduced into the host cell and related to the targeted enzyme, ie, fucose modification.
  • a host cell for preparing the haptoglobin composition of the present invention can be prepared.
  • a yeast cell As a host cell, a yeast cell, an animal cell, an insect cell, a plant cell, etc. can be used as long as it has a gene for an enzyme related to the target fucose modification.
  • the recombinant vector introduction method suitable for various host cells described in 3 below can be used.
  • Examples of the method for preparing cDNA of the enzyme related to fucose modification include the method for preparing cDNA described in (1) (a) of 1 above.
  • Examples of a method for preparing genomic DNA of an enzyme related to fucose modification include the method for preparing genomic DNA described in (1) (a) of 1 above.
  • the DNA base sequence is cleaved with an appropriate restriction enzyme, and then subcloned into a plasmid such as pBluescript SK (-) (Stratagene), and a commonly used base sequence analysis method such as Sanger ( Sanger) et al. [Procedures of the National Academia Sci., USA), 74, 5463 (1977)] This can be confirmed by analysis using an automatic base sequence analyzer, for example, a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems).
  • a base sequence analyzer such as ABI PRISM377 DNA Sequencer (Applied Biosystems).
  • RDO can be prepared by a conventional method or using a DNA synthesizer.
  • RDO constructs are described in Science, 273, 1386 (1996); Nichiya's Medicine (Nature Medicine), 4, 285 (1998); Hepatology, 25, 1462 (1997); Gene 'Therapies (Gene Therapy), 5, 1960 (1999); Gene' Therapies (Gene Therapy), 5, 1960 (1999); Journal 'Ob' Molekiyura 'Medellin (J. Mol. Med.), 75,829 (1997); Procedures' Ob The National 'Academy ⁇ ⁇ ⁇ Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 8774 (1999); The National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci.
  • the host cell used for preparing the haptoglobin composition of the present invention can be prepared as follows, for example, by targeting the gene of an enzyme related to fucose modification and using the RNAi method.
  • cDNA is prepared using the method described in (1) (a) of 1 above of an enzyme related to fucose modification.
  • RNAi gene construct of an appropriate length that includes the enzyme coding for fucose modification or the untranslated region is designed.
  • RNAi gene in order to express the RNAi gene in a cell, a prepared cDNA fragment or full length Is inserted downstream of the promoter of an appropriate expression vector to prepare a thread-replacement vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • the haploglobin composition of the present invention by selecting a transformant using as an index the activity of the enzyme related to the introduced fucose modification, the glycoprotein molecule produced or the sugar chain structure of the glycoprotein on the cell surface.
  • the host cell to be used can be obtained.
  • any yeast cell, animal cell, insect cell, plant cell, etc. having an enzyme gene related to the target fucose modification can be used.
  • RNAi gene a vector that can replicate autonomously in the host cell or can be integrated into a chromosome and contains a promoter at a position where the designed RNAi gene can be transcribed is used.
  • the expression vector described in 3 below can be mentioned.
  • Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) in this section 1.
  • Examples include the method for preparing cDNA described in (a) of 1).
  • the host cell used for preparing the haptoglobin composition of the present invention can be obtained by directly introducing into the host cell the RNAi gene designed based on the base sequence of the enzyme related to fucose modification without using an expression vector. It can also be obtained.
  • RNAi gene can be prepared by a conventional method or using a DNA synthesizer.
  • NAi gene The construct of the NAi gene is [Nature, 391, 806 (1998); Gus Ob The National National Power Science (Proc.Natl. Acad. Sci. USA), 95, 15502 (1998); Nature, 395, 854 (1998); Proc Ding's 'Ob' The • National 'A Power Demi-Ob' Science (Proc. Natl. Acad. Sci. USA), 96, 5049 (19 99); Cell,, 1017 (1998); Proc Deings' Ob The National 'A Power Demi ⁇ Ob' Science (Proc. Natl. Acad. Sci.
  • the host cell used for producing the haptoglobin composition of the present invention is a transposon system described in Nature Genet., 25, 35 (2000), etc.
  • a host cell used for preparing the haptoglobin composition of the present invention can be prepared by selecting a mutant using as an index the activity or the glycoprotein structure of the glycoprotein molecule or glycoprotein on the cell membrane. .
  • the transposon system is a system that induces mutations by randomly inserting foreign genes onto the chromosome, and is usually used as a vector to induce mutations in foreign genes inserted into transposons.
  • a transposase expression vector for randomly inserting the gene into the chromosome is introduced into the cell at the same time.
  • a transposase can be used if it is suitable for the transposon sequence used!
  • any gene can be used as long as it induces a mutation in the DNA of the host cell.
  • any yeast cell, animal cell, insect cell, plant cell or the like having an enzyme gene related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • the method for introducing recombinant vectors suitable for various host cells described in 3 below is used. You can!
  • Examples of a method for selecting a mutant using as an index the activity of an enzyme related to fucose modification include the method described in (a) of (1) of this section 1.
  • Examples of a method for selecting a mutant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (5) of this section 1. Examples of the method for selecting a mutant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • the host cell used to produce the haptoglobin composition of the present invention is related to fucose modification.
  • enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx.
  • N-glycoside bond As an enzyme involved in the sugar chain modification in which the 1-position of fucose is a-linked to the 6-position of the N-acetylyldarcosamine at the reducing end of the complex sugar chain, ⁇ ⁇ , 6— Examples include fucosyltransferase and ⁇ -L-fucosidase.
  • These enzymes are enzymes that catalyze a specific reaction having substrate specificity, and these enzymes are destroyed by destroying the active center of the catalytic activity having substrate specificity. A dominant negative form of the enzyme can be prepared. G of target enzymes
  • GMD derived from CHO cells (SEQ ID NO: 8)
  • 155th threonine 157th glutamic acid
  • 179th tyrosine 183th
  • lysine a dominant negative
  • Genes with such amino acid substitutions were prepared using site-directed mutagenesis described in Molecular and Cloning 2nd Edition, Current Protocols' In Molecular ⁇ ⁇ Biology etc. Can be done.
  • the host cell used to produce the haptoglobin filamentous product of the present invention contains a gene encoding a dominant negative form of the target enzyme produced as described above (hereinafter abbreviated as a dominant negative form gene).
  • a dominant negative form gene a gene encoding a dominant negative form of the target enzyme produced as described above.
  • a dominant negative gene of an enzyme related to fucose modification is prepared.
  • a DNA fragment of an appropriate length containing a portion encoding the protein is prepared.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length DNA downstream of the promoter of an appropriate expression vector.
  • a transformant is obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • any yeast cell, animal cell, insect cell, plant cell, etc. having a gene for an enzyme related to the target fucose modification can be used.
  • the host cells described in 3 below can be mentioned.
  • an expression vector autonomous replication is possible in the above host cell or into the chromosome.
  • the one containing a promoter at a position where the DNA encoding the target dominant negative can be transcribed is used.
  • the expression vector described in 3 below can be mentioned.
  • Examples of the method for selecting a transformant using the activity of an enzyme related to fucose modification as an index include the method described in (a) of (1) below.
  • Examples of the method for selecting a transformant using the sugar chain structure of a glycoprotein on a cell membrane as an index include the method described in (1) below. Examples of the method for selecting a transformant using the sugar chain structure of the produced glycoprotein molecule as an index include the methods described in 5 and 6 below.
  • the host cell used to prepare the haptoglobin composition of the present invention uses a technique in which a mutation is introduced into a gene of an enzyme related to fucose modification and a desired cell line in which the enzyme is mutated is selected. Can be produced.
  • enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx.
  • Specific examples of the enzyme involved in the sugar chain modification in which the 1-position of fucose is ⁇ - linked to the 6-position of N-glycidyl glucosamine at the N-glycoside-linked complex sugar chain reducing terminal include ⁇ ⁇ , 6-fucosyltransferase, Examples include ⁇ -L-fucosidase.
  • a method for introducing a mutation into an enzyme related to fucose modification 1) It is related to fucose modification from a mutant in which a parent strain was treated by a mutagenesis treatment or a naturally occurring mutant. 2) A method for selecting a desired cell line based on the activity of the enzyme to be produced, 2) a sugar chain of a production glycoprotein molecule from a mutant obtained by treating the parent strain by mutagenesis treatment or a spontaneously generated mutant. A method of selecting a desired cell line using the structure as an index; 3) a glycoprotein sugar on the cell membrane of a cell derived from a mutant obtained by treating a parent line by mutagenesis treatment or a naturally occurring mutant. Examples thereof include a method of selecting a desired cell line using the chain structure as an index. [0111] As the mutagenesis treatment, there are point mutations and deletions in the DNA of the parent cell line.
  • Any treatment that induces a mutation can be used.
  • mutagenic agent treatments with ethyl nitrosourea, nitrosoguanidine, benzopyrene, and atalidine dye, and irradiation with radiation.
  • Various alkylating agents and carcinogens can also be used as mutagens. Examples of methods for causing a mutagenic agent to act on cells include, for example, tissue culture technology 3rd edition (Asakura Shoten) edited by the Japanese Society for Tissue Culture (1996), Nature Genet., 24, 314, (2000) and the like.
  • Spontaneously occurring mutants include sudden mutants that occur spontaneously by continuing subculture under normal cell culture conditions without special mutagenesis. be able to.
  • Examples of the method for identifying the sugar chain structure of the produced glycoprotein molecule include the methods described in 5 and 6 below.
  • Examples of the method for identifying the sugar chain structure of a glycoprotein on the cell membrane include the method described in 1 (5) of this section.
  • the host cell used to produce the haptoglobin composition of the present invention targets the gene of an enzyme related to fucose modification, and antisense RNAZDNA technology [Bioscience and Industry, 50, 322 (1992), Chemistry, 681 (1991), Biotechnology, 9,358 (1992), Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology,! ⁇ , 152 (1992), Cell engineering, 16, 1463 (1997)], Triple helix technology [ Trends in Biotechnology, 10, 1 32 (1992)] or the like, and can be produced by suppressing transcription or translation of the target gene.
  • enzymes involved in the synthesis of intracellular sugar nucleotide GDP-fucose include GMD and Fx.
  • an enzyme involved in the sugar chain modification in which the 1-position of fucose is ⁇ - bonded to the 6-position of N-glycidylcolcamine at the N-glycosidic complex sugar chain reducing terminal Specific examples include ⁇ , 6-fucosyltransferase, ⁇ -L-fucosidase, and the like.
  • the host cell used to prepare the haptoglobin composition of the present invention is a lectin that recognizes a sugar chain structure in which the N-glycidyl-linked sugar chain reducing terminal N-acetylylcosamine 6-position and fucose 1-position are linked. It can be produced by using a technique for selecting strains that are resistant to.
  • any lectin can be used as long as it recognizes a sugar chain structure in which the N-glycidylcolcamine 6-position of the N-glycoside-linked sugar chain reducing end and the 1-position of fucose are a- linked.
  • Specific examples include Lentil lectin LCA (Lentil Agglutinin derived from Lg Culinaris) Endumerme lectin PSA (Peum sativum derived Pe a Lectin), Broad bean lectin VFA (Agglutinin derived from Viciafaba), Hirochawantake lectin AAL ( Lectin from Aleuria aurantia).
  • the cells are cultured in a medium containing the above-mentioned lectin at a concentration of 1 ⁇ g / mL to 1 mg / mL for 1 to 2 weeks, preferably 1 to 1 week, and surviving cells are passed. Subculture or colonies are picked up, transferred to another culture vessel, and further cultured in a medium containing lectin, whereby the N-glycidyl-linked sugar chain reducing terminal N-acetylyldarcosamine of the present invention and position 1 of fucose A strain resistant to a lectin that recognizes an a-linked sugar chain structure can be selected.
  • Transgenic non-human animals, plants, or their progeny whose genomic genes have been modified so that the enzyme activity involved in the modification of the sugar chain of the haptoglobin molecule is controlled can synthesize intracellular sugar nucleotides GDP-fucose.
  • Enzyme involved or N-glycoside bond complex The embryonic nature of the present invention produced using the above 1 targeting the gene of the enzyme involved in glycosylation in which the 1-position of fucose is ⁇ -linked to the 6-position of N-acetylyldarcosamine at the reducing end of the combined sugar chain
  • it can be prepared from stem cells, fertilized egg cells, and plant callus cells as follows.
  • target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, and embryonic stem cells such as rabbits, monkeys, and rabbits.
  • target non-human animals such as ushi, hidge, goats, pigs, horses, mice, rats, and embryonic stem cells such as rabbits, monkeys, and rabbits.
  • embryonic stem cell clones and normal cell clones can be obtained by methods such as injection chimera or assembly chimera into fertilized eggs of animals.
  • a chimeric individual can be prepared. By combining this chimeric individual with a normal individual, the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose in whole body cells or the N-glycidyl-linked N-acetylyldarcosamine 6
  • Transgenic non-human animals can be obtained in which the activity of the enzyme involved in sugar chain modification in which position 1 of fucose is a- linked is reduced.
  • fertilized egg cells such as ushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey, and rabbit
  • the activity of the enzyme involved in the synthesis of intracellular sugar nucleotides GDP-fucose or the 1-position of fucose at the 6-position of N-glycyl-linked N-acetylyldarcosamine at the N-glycoside-linked complex type sugar chain reducing end A fertilized egg cell of the present invention in which the activity of an enzyme involved in modification of an oc-linked sugar chain is reduced can be produced.
  • the fertilized egg cells thus produced are transplanted into the oviduct or uterus of a pseudopregnant female using the embryo transfer method described in Mupureating 'Mouse' Embryo 2nd edition, etc. Nucleotide GDP-enzyme activity involved in the synthesis of fucose or N-glycoside-linked complex sugar chain-reducing terminal N-acetylyldarcosamine Transgenic non-human animals with reduced activity can be produced.
  • the enzyme involved in the synthesis of intracellular sugar nucleotide GDP-fucose can be obtained by using the same method as described in 1. above on the target plant strength or cells.
  • To produce a callus of the present invention in which the activity of the enzyme involved in the sugar chain modification in which the 1-position of fucose is a- linked to the 6-position of the N-glycidalcosamine at the reducing end of the active or N-glycoside-linked complex type sugar chain is reduced Can do.
  • the prepared callus was prepared by a known method [tissue culture, 20 (1994); tissue culture, 21 (1995);
  • the cells are cultured again in a medium containing auxin and cytokinin to synthesize intracellular sugar nucleotides GDP-fucose.
  • the haptoglobin composition of the present invention is a molecular 'cloning second edition, current' protocorores in molecular neurology, Antibodies, ALaboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter abbreviated as antibodies), Monoclonal Antibod ies: pnnciples and practice, Third Edition, Acaa.Press, 1993 (below, Monochrome ⁇ Nanore Antibodys), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter referred to as Antibody Engineering) For example, it can be obtained by expressing in a host cell as follows.
  • a full-length cDNA of HP-1 type or HP-2 type haptoglobin molecule was prepared, and the haptoglobin was prepared. Prepare an appropriate length of DNA fragment containing the molecule-encoding portion.
  • a recombinant vector is prepared by inserting the DNA fragment or full-length cDNA into the downstream of the promoter of an appropriate expression vector.
  • a transformant producing a haptoglobin molecule can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
  • any yeast cell, animal cell, insect cell, plant cell, etc. that can express the target gene can be used.
  • Cells obtained by various artificial techniques shown in 1 can also be used as host cells.
  • an expression vector one that can replicate autonomously in the above host cell or can be integrated into a chromosome and contains a promoter at a position where the DNA encoding the target haptoglobin molecule can be transcribed is used. It is done.
  • the cDNA is obtained from a human or non-human animal thread or tissue or a cell-specific probe or primer. Etc. can be used.
  • yeast When yeast is used as a host cell, examples of expression vectors include YEP13 (ATC C37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like. Any promoter can be used as long as it can be expressed in yeast strains. For example, promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Gall promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter and the like.
  • promoters of glycolytic genes such as hexose kinase, PH05 promoter, PGK promoter, GAP promoter, ADH promoter Gall promoter, gal 10 promoter, heat shock protein promoter, MF al promoter, CUP 1 promoter and the like.
  • Examples of host cells include microorganisms belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybe mouth Mrs, Trichosporon, Schusomyces, etc. You can power chwanniomvces alluvius.
  • a method for introducing the recombinant vector if the method is to introduce DNA into yeast, the deviation is also used.
  • an animal cell is used as a host, as an expression vector, for example, pcDNAU pcD M8
  • Any promoter can be used as long as it can be expressed in animal cells.
  • a promoter of the cytomegalovirus (CMV) IE (immediateearly) gene an early promoter of SV40, a retroinores promoter , Meta-mouthone promoter, heat shock promoter, SRa promoter, and the like.
  • CMV cytomegalovirus
  • any method can be used as long as it is a method for introducing DNA into animal cells.
  • the electoral position method [Cytote chnology, 3, 133 (1990)]
  • Calcium phosphate method [Japanese Patent Laid-open No. 2-227075]
  • Lipofection method [Proceedings of the National Academy of Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)]
  • injection method [Map of 'The Mouse' Embryo Laboratory Laboratory Manual]
  • method using particle gun (gene gun) [Patent No. 2606856 , Patent No.
  • the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
  • Examples of the gene transfer vector used in the method include pVL1392, pVLl393, pBlueBacIII (both Invitorogen) and the like.
  • Autographa californica nu clear polyhedrosis virus can be used for the outgrafa 'Cali forum-force' Nuclea 1 'polyhedrosis' virus, which is a virus that infects the night stealing insects.
  • Insect cells include ovarian cells of Spodoptera frugiperda, S19, S1 1 [Current 'Pokoto Reno' In 'Molechu Fu'Noroshi 1 Baculovirus Expression Vectors, A La boratory Manual, WH Freeman and Company, New York (1992)], Trichoplusiani ovary cells such as High 5 (Invitrogen), etc. can be used.
  • Examples of methods for co-introducing the above recombinant gene transfer vector and the above baculovirus into insect cells for preparing recombinant viruses include, for example, the calcium phosphate method (JP-A-2-227075), the lipofusion method [Proceedings' ⁇ The 'National' Academia's Science (Pro Natl. Acad. Sci. USA), 84, 7413 (1987)].
  • expression vectors When plant cells are used as host cells, examples of expression vectors include Ti plus Examples include midos and tobacco mosaic virus vectors.
  • Any promoter can be used as long as it can be expressed in plant cells, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the rice 1 promoter.
  • CaMV cauliflower mosaic virus
  • host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.
  • any method can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium [April 59-140]
  • the haptoglobin composition can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating haptoglobin molecules in the culture, and collecting the haptoglobin from the culture.
  • the method of culturing the transformant in a medium can be performed according to a usual method used for culturing host cells.
  • a medium for culturing a transformant obtained by using a eukaryote such as yeast as a host it contains a carbon source, a nitrogen source, inorganic salts, etc. that can be assimilated by the organism, so that the transformant can be cultured efficiently. If the medium can be used, the difference between natural and synthetic media can be used.
  • the carbon source as long as the organism can assimilate, glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolyzate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate.
  • Ammonium salts of inorganic or organic acids such as humic and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, Soybean koji and soybean koji hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride salt, ferrous sulfate, mangan sulfate, copper sulfate, calcium carbonate, etc. are used. be able to.
  • the culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is 15-40 ° C, and the culture time is usually 16 hours to 7 days.
  • the pH during the culture is maintained at 3.0 to 9.0.
  • the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the medium as necessary.
  • an inducer may be added to the culture medium.
  • RPMI 1640 medium commonly used as a medium for culturing transformants obtained using animal cells as a host [The Journal of the American American Medical Association (The Journal of the American Medical Association), Plastic, 519 (1967)], Eagle's MEM medium [Science, 12 ⁇ , 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 ( 1959), 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], Whitten medium [Developmental engineering] Experiment Manual-Transgeneic • How to make mice (Kodansha) Motoya Katsaki (1987) Hota can use media such as fetal calf serum added to these media. [0144] Cultivation is usually carried out for 1 to 7 days under conditions of pH 6-8, 30-40 ° C, 5% CO, etc.
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during culture.
  • Cultivation is usually carried out under conditions of pH 6-7, 25-30 ° C, etc. for 1-5 days.
  • antibiotics such as gentamicin may be added to the medium as needed during the culture.
  • Transformants obtained using plant cells as hosts are cultured as cells or differentiated into plant cells and organs. can do.
  • a medium for culturing the transformant commonly used Murashige 'and' Stag (MS) medium, White medium, or a plant hormone such as auxin or cytokinin is added to these mediums. It is possible to use the prepared medium.
  • the culture is usually carried out under conditions of pH 5-9 and 20-40 ° C for 3-60 days.
  • antibiotics such as kanamycin and hygromycin may be added to the medium as needed during the culture.
  • a transformant derived from a microorganism, animal cell, or plant cell having a thread-recombinant vector in which DNA encoding a haptoglobin molecule is incorporated is cultured according to a normal culture method, and a haptoglobin composition is obtained.
  • a haptoglobin composition can be produced by producing and accumulating products and collecting the haptoglobin composition from the culture.
  • the haptoglobin composition can be produced in a host cell, secreted outside the host cell, or produced on the host cell membrane.
  • the method can be selected by changing the structure of the haptoglobin molecule to be generated.
  • DNA encoding a haptoglobin molecule and DNA encoding a signal peptide suitable for expression of the haptoglobin molecule are inserted into an expression vector, and the expression vector is inserted into a host cell.
  • the target haptoglobin molecule can be actively secreted outside the host cell.
  • the production amount can also be increased by using a gene amplification system using.
  • an animal individual transgenic non-human animal
  • a plant individual transgenic plant
  • a haptoglobin composition can also be produced using.
  • the transformant is an animal individual or a plant individual, it is reared or cultivated according to a usual method, and a haptoglobin composition is produced and accumulated, and the haptoglobin composition is collected from the animal individual or plant individual.
  • the hubtoglobin composition can be produced.
  • a method for producing a haptoglobin composition using an animal individual for example, a known method [American Journal of Clinical Nutrition, 63, 639S (1996); American Introduced gene according to Journal of Clinical Nutrition, 63, 627S (1996); Bio / Technology, 9, 830 (1991)] Created
  • a method for producing a target haptoglobin composition in an animal In the case of an individual animal, for example, a transgenic non-human animal introduced with DNA encoding a haptoglobin molecule ⁇ ! Is generated and accumulated in the animal, and the haptoglobin composition is produced from the animal.
  • a haptoglobin composition can be produced by collecting the product. Examples of the production / accumulation location in the animal include milk of the animal (Japanese Patent Laid-Open No. 63-309192) and eggs.
  • Any promoter can be used as long as it can be expressed in animals. For example, a casein promoter, 13 casein promoter, j8 lactoglobulin promoter, whey acid, which are mammary cell specific promoters. (3) A protein promoter or the like is preferably used.
  • a method for producing a haptoglobin composition using an individual plant for example, a known method for transgenic plants into which DNA encoding a ptoglobin molecule has been introduced [tissue culture, (1994); tissue culture] , 21 (1995); cultivated according to Trends “In” biotechnology (Trends in Biotec hnology), 15, 45 (1997)], and a haptoglobin composition is produced and accumulated in the plant.
  • a method for producing a haptoglobin composition by collecting the composition can be mentioned.
  • a hub globin composition produced by a transformant introduced with a gene encoding a haptoglobin molecule is expressed in a dissolved state in cells
  • the cell is centrifuged after completion of the culture. Collected by separation, suspended in an aqueous buffer solution, and then disrupted with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as Jetylaminoethyl (DEAE) -Sepharose, DIA IONHPA-75 (Mitsubishi Chemical Corporation), 3 ⁇ 4- Sepharose F (Pharmacia
  • a purified preparation of the composition can be obtained. Specifically, a method combining PEG fractionation, ammonium sulfate fractionation, and ion-exchange chromatography can be mentioned (development of follow-up medicines 1
  • the haptoglobin composition When the haptoglobin composition is expressed by forming an insoluble substance in the cells, the cells are similarly collected, disrupted, and centrifuged to obtain an insoluble substance of the haptoglobin composition as a precipitate fraction. to recover.
  • the recovered insoluble material of the haptoglobin composition is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution, the hubtoglobin composition is returned to a normal three-dimensional structure, and then a purified sample of the hubtoglobin composition can be obtained by the same isolation and purification method as described above. .
  • the habutoglobin composition or a derivative thereof can be recovered in the culture supernatant. That is, the culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and the haptoglobin composition is obtained from the culture supernatant by using the same isolation and purification method as described above. It is possible to obtain a purified sample.
  • the haptoglobin composition of the present invention can be produced by purifying the haptoglobin composition.
  • the biological activity of the purified nobutoglobin composition can be measured using various known methods. Specifically, a method for measuring hemoglobin binding activity, a method for measuring CD163 binding activity which is a complex of hemoglobin and haptoglobin (hereinafter abbreviated as hemoglobin haptoglobin complex), a method for measuring intracellular signal from CD163, CD163
  • hemoglobin haptoglobin complex a method for measuring intracellular signal from CD163, CD163
  • In vitro tests such as interleukin and granulocyte-macrophage colony-stimulating factor production measurement method, plastaglandin synthesis inhibition activity measurement method, angiogenic activity measurement method, antibacterial activity measurement method, etc.
  • In vivo tests or clinical tests using humans Clin. Chem., 42, 1589 (1996), Eur. J. Clin • Chem.
  • Paokishidase activity of hemoglobin is suppressed at a low P H condition.
  • hemoglobin combined with haptoglobin can exhibit peroxidase activity even under low pH conditions.
  • a reagent kit for measuring the binding amount of haptoglobin and hemoglobin using this property is commercially available (manufactured by Tridelta). Hemoglobin binding activity can be quantified by carrying out this reagent kit according to the attached operation procedure manual.
  • Phosphate buffer solution pH7.0 manufactured by Invitrogen
  • PBS Phosphate buffer solution pH7.0
  • soluble CD163 J. Biol. Chem., 279, 51 561 (2004)
  • PBS Phosphate buffer solution pH7.0
  • the absorbance in the reaction solution was measured at a wavelength of 415, and the test substance haptoglobin was added, and the reaction solution in which the test substance or standard product at each dilution stage was added from the absorbance of the control reaction solution.
  • a value obtained by subtracting the absorbance of. This value is plotted on a semilogarithmic graph with the vertical axis representing the amount of haptoglobin bound to the neuroglobin receptor and the horizontal axis representing the dilution ratio of the test substance or standard.
  • the standard of the test substance By approximating the relationship between the amount of haptoglobin bound to the haptoglobin receptor and the dilution rate from the plotted measurement values, and comparing the approximate equations obtained as a result of measurement of the test substance and the standard product, the standard of the test substance The magnification for the product can be determined and its titer can be determined.
  • the binding activity between solubilized CD163 and hemoglobin haptoglobin complex should be measured using the surface plasmon resonance phenomenon by the method described in the literature [J. Biol. Chem., 279, 51561 (2004)]. You can also.
  • CD163 binding activity assay (method using CD163 expressing cells)
  • a purified haptoglobin composition as a test substance and a commercially available habutoglobin with a known concentration and specific activity as a standard product using PBS containing ushi serum albumin at a volume ratio of 1%, a certain amount Of hemoglobin.
  • This hemoglobin haptoglobin complex is added to cells expressing CD163 and incubated at 4 ° C for 1 hour or longer.
  • Rush serum albumin containing 1% volume ratio After rinsing cells several times with PBS to remove the hemoglobin haptoglobin complex that did not bind to the cell surface, it contained ushi serum albumin at a volume ratio of 1%.
  • the biological activity of haptoglobin is measured by measuring an increase in calcium concentration, an increase in inositol triphosphate concentration, or an increase in production of interleukin 1, interoral quinine 6, or granulocyte-macrophage colony-stimulating factor. (J. Leukoe. Biol, 66, 8 58 (1999); Circ. Res., 92, 1193 (2003)).
  • a plataglandin synthesis caused by hemoglobin is inhibited by binding a hemoglobin composition purified as a test substance to hemoglobin.
  • the biological activity of haptoglobin can be measured (Anthropol. Anz., 50, 281 (1992)).
  • mouse globin By adding a purified rabbit globin composition as a test substance to vascular epithelial cells, it is possible to observe angiogenic activities such as cell proliferation and cell proliferation, and to increase the strength of the vascular new life.
  • the biological activity of mouse globin can be measured by measuring (J. Clin. Invest "91, 977 (1993)).
  • a clearance test using a purified rabbit globin composition or the like can be examined using a model animal such as a rat.
  • a test substance that also has a haptoglobin composition or a haptoglobin-hemoglobin complex and a test substance that has been labeled with a radioisotope by a known method is administered to a rat by intravenous injection or subcutaneous injection.
  • Samples in rat blood can be collected by collecting blood at any time after administration and measuring the amount of the labeled compound or ELISA method that can specifically detect and quantify human hubtoglobin.
  • the concentration The amount of labeling of the test substance that has been labeled is measured on the label. Measurement is performed by a known analysis method. The obtained data is used to measure the pharmacokinetic parameters of each rat using, for example, PCNONLIN nonlinear regression analysis (Statistical Consultants, 1992).
  • the clearance test for haptoglobin compositions can also be evaluated using animal models such as primates that are more closely related to humans than rats.
  • the sugar chain structure of a haptoglobin molecule expressed in various cells can be performed according to the analysis of the sugar chain structure of a normal glycoprotein.
  • sugar chains bound to haptoglobin molecules are composed of neutral sugars such as galactose and mannose, amino sugars such as N-acetyldarcosamine, and acidic sugars such as sialic acid. It can be carried out by using a method such as sugar chain structure analysis using a sugar chain map method.
  • neutral sugar or amino sugar can be liberated by acid hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
  • composition ratio can also be analyzed by a fluorescent labeling method using 2-aminoviridine. Specifically, a sample obtained by acid-branching decomposition according to a known method [Agric. Biol. Chem., 55 (1). 283-284 (1991)] -Fluorescent labeling by aminobilylation and composition analysis can be calculated by HPLC analysis.
  • the two-dimensional glycan mapping method is, for example, the retention time or the glycan retention time by reverse phase chromatography on the X axis. Is a method of estimating the sugar chain structure by plotting the elution position and the retention time or elution position of the sugar chain by normal phase chromatography on the Y axis and comparing them with the results of known sugar chains. is there.
  • the haptoglobin composition was decomposed with hydrazine, the haptoglobin molecular force also released the sugar chain, and fluorescent labeling of the sugar chain with 2-aminoviridine (hereinafter abbreviated as “PA”) [Journal After performing “Biochemistry” (J. Biochem.), 197 (1984)], the glycan is separated from excess PA reagent by gel filtration and subjected to reverse phase chromatography. Next, normal phase chromatography is performed for each peak of the collected sugar chain.
  • PA 2-aminoviridine
  • mass analysis such as MALDI-TOF-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain map method.
  • the haptoglobin composition is composed of haptoglobin molecular forces having different sugar chain structures.
  • the haptoglobin composition of the present invention is characterized in that fucose is not bound to ⁇ -acetyldarcosamine at the reducing end of ⁇ -glycoside bond complex type sugar chain, and has a long blood half-life.
  • Such a haptoglobin composition can be identified by using the method for analyzing the sugar chain structure of the haptoglobin molecule described in 5. above. It can also be identified by using an immunological quantification method using lectins.
  • lectins used for identifying the sugar chain structure of haptoglobin molecules include WG A (wneat— germ agglutinin derived from T. vulgaris) onA (concanavali n A derived from C. ensiformis), RIC (derived from R. communis) ), L- PHA (leukoagglutinin from P. vulgaris), LC A (lentil agglutinin from L. culinaris), PSA (Pea lectin from P.
  • AAL Alpha-1 (Aleuanthria caustus Lectin) ) ⁇ BPL (Bauhinia purpurea Le ctin) 8 DSL (Datura stramonium Lectin) ⁇ DBA (Dolichos biflorus Agglutinin) ⁇ EBL (E1 derberry Balk Lectin) ⁇ ECL (Erythrina cristagalli Lectin) ⁇ EEL (Euthymus europaeu s Lectin), GNL (Galanth Lectin), GSL (Griffonia simphcifolia Lectin), HPA (Helix pomatia Agglutinin) ⁇ HHL (Hippeastrum Hybrid Lectin) ⁇ Jacalin, LTL (Lotus t etragonolobus Lectin), LEL (Lycopersicon esculentum Lectin), MAL (Maackia am
  • a lectin that specifically recognizes a sugar chain structure in which fucose is bound to N-acetylcolcamine at the N-darcoside-linked complex type sugar chain reducing end examples include lentil lectin LCA (Lentil Agglutinin from Lens Culinaris), endangered lectin PSA (Peasum sativum-derived Pea Lectin), broad bean lectin VFA (Agglutinin from Viciafaba), An example is chiatake lectin AAL (Lectin derived from Aleuria aurantia).
  • the haptoglobin composition of the present invention has a half-life in blood when administered in vivo, compared to haptoglobin in which fucose is bound to N-glycidyl darcosamine at the N-glycoside-linked complex type sugar chain reducing end. It has the characteristic of being long. Therefore, the haptoglobi of the present invention
  • the composition can enable treatment with fewer doses and fewer doses in various diseases for which treatment with haptoglobin is indicated. This not only reduces the burden on patients and medical sites, but also contributes to the reduction of accidents and side effects resulting from current treatments that must be administered in large quantities.
  • haptoglobin preparations are produced from the plasma of multiple healthy individuals, various hepatitis viruses, AIDS viruses, human parvoviruses, mutant Creutzfeld's prions that cause Jacob disease, etc. The fear of infection cannot be completely denied. It is very useful to switch a currently marketed blood product to a product containing the haptoglobin composition of the present invention in order to prevent various infection accidents. Furthermore, polymorphisms and multimers exist in haptoglobin, and its physiological function has been reported to be different for each polymorphism or multimer. By using the haptoglobin composition of the present invention, it can be adapted to the indication disease. It is possible to prepare a therapeutic agent containing a suitable haptoglobin type and provide it to the patient.
  • Diseases using the haptoglobin composition of the present invention are also effective for the prevention and treatment of diseases involving hemolysis, diseases involving tissue damage caused by blood free hemoglobin, and bacterial infections.
  • hemolytic diseases Specifically, traumatic hemoglobinuria associated with burns and blood transfusions, open-heart surgery under extracorporeal circulation such as cardiopulmonary bypass, and functional damage to the liver, the organ that produces haptoglobin Alcoholic hepatitis or alcoholic cirrhosis, congenital deficiency, etc.
  • pathological symptoms such as hemoglobinuria and hemoglobinemia are exhibited, and anemia associated with the outflow of iron and renal damage due to adhesion of hemoglobin and iron to the kidney are observed.
  • Examples of diseases associated with yarn and tissue injury caused by blood free hemoglobin include diseases associated with oxidative vascular injury caused by blood free hemoglobin, diseases associated with renal tubule injury caused by blood free hemoglobin, and the like.
  • Diseases associated with acid-induced vascular injury due to blood free hemoglobin include diseases caused by the formation of oxidized LDL and oxidative damage of vascular endothelial cells, diseases exacerbated by free radical generation, and diseases caused by acid oxidation of nucleic acids. can give.
  • diseases caused by the formation of oxidized LDL and acid dysfunction of vascular endothelial cells include myocardial infarction, cerebral infarction, and arteriosclerosis (American Journal of Medicine, 105, 32S (1998)).
  • Diseases exacerbated by the generation of free radicals include emphysema, pulmonary fibrosis, acute respiratory distress syndrome, neurodegenerative diseases, and chronic inflammatory diseases.
  • diseases caused by acid oxidation of nucleic acids include carcinogenesis.
  • Examples of diseases associated with renal tubule injury caused by blood free hemoglobin include nephrotic syndrome, acute nephritis, and chronic nephritis.
  • Arteriosclerosis is a disease in which blood flow is impaired due to narrowing of the arterial vascular lumen, and arteries that have lost the elasticity of the arterial wall cause dilation, rupture, and the like.
  • Hpl type haptoglobin the effect of anti-acidic vitamins such as vitamins C and E has been observed to prevent arteriosclerosis (Diabetes Care, 27, 925 (2004)).
  • Myocardial infarction is a disease in which myocardial ischemia occurs due to vascular injury due to occlusion or stenosis of the coronary artery that sends blood to the heart, resulting in necrosis of the myocardium.
  • myocardial ischemia occurs due to vascular injury due to occlusion or stenosis of the coronary artery that sends blood to the heart, resulting in necrosis of the myocardium.
  • Hp2 haptoglobin gene type Circ. Res., 92, 1193 (2003).
  • Cerebral infarction is a disease in which cerebral ischemia occurs due to vascular injury caused by occlusion or stenosis of blood vessels that send blood to the brain, resulting in necrosis of brain cells.
  • Emphysema is a disease in which the alveolar wall is destroyed, and the adjacent alveoli are fused together to form cavities, reducing the surface area of the alveoli. A reduction in alveolar surface area leads to a decrease in lung function and induces significant dyspnea.
  • Pulmonary fibrosis is a pneumonia disease in which inflammation occurs in the lung stroma such as the alveolar wall.
  • the pharmaceutical containing the haptoglobin composition of the present invention can be administered alone as a prophylactic or therapeutic agent. Usually mixed with one or more pharmacologically acceptable carriers. However, it is desirable to provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
  • the route of administration includes oral administration, where it is desirable to use the most effective treatment, or parenteral administration, such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous.
  • parenteral administration such as buccal, respiratory tract, rectal, subcutaneous, intramuscular and intravenous.
  • intravenous administration can be desirable
  • the dosage form include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups include water, sucrose, sorbitol, fructose, etc., Daricols such as polyethylene glycol, propylene glycol, oils such as sesame oil, olive oil, soybean oil, P-hydroxy Preservatives such as benzoates, strawberry
  • Flavors such as laver and peppermint can be used as additives.
  • Capsules, tablets, powders, granules and the like are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc It can be produced using a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • Suitable preparations for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both.
  • a powder injection can be prepared by lyophilizing the haptoglobin composition of the present invention according to a conventional method and adding sodium chloride salt thereto.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant does not irritate the hubtoglobin composition itself, or a carrier that does not irritate the recipient's oral cavity and airway mucosa and facilitates absorption by dispersing the habutoglobin composition as fine particles. It is prepared using.
  • the carrier include lactose and glycerin.
  • the carrier include lactose and glycerin.
  • aerosols, dry powders and the like it is possible to prepare aerosols, dry powders and the like.
  • ingredients exemplified as additives for oral preparations to these parenteral preparations.
  • the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but the amount of active ingredient is usually 4000 units per adult (1 unit is 1 mg The amount that can bind to hemoglobin of about 5.7 g).
  • the methods for examining the biological activity of the haptoglobin composition can be determined from the above-described hemoglobin binding activity measurement method, the CD163 binding activity measurement method for the hemoglobin haptoglobin complex receptor, and CD163. Intracellular signal measurement method through CD163
  • Examples include an in vitro test such as a measurement method for langine synthesis inhibitory activity, a method for measuring angiogenic activity, a method for measuring an antibacterial activity, or an in vivo test using a hemolyzed model animal.
  • an in vitro test such as a measurement method for langine synthesis inhibitory activity, a method for measuring angiogenic activity, a method for measuring an antibacterial activity, or an in vivo test using a hemolyzed model animal.
  • a FUT8 gene double knockout cell line producing human HP-1 haptoglobin was prepared by the method shown below.
  • the solution after the PCR reaction was subjected to 2% agarose gel electrophoresis, and an approximately 1067 bp PCR amplified fragment (containing a noptoglobin HP-1 type cDNA sequence) was purified using QIAquick Gel Extraction kit (manufactured by QIAGEN).
  • the obtained purified DNA fragment was dissolved in 17 L of water. Thereafter, 7.5 L of restriction enzyme EcoRI (manufactured by Takara Bio Inc.), 5 units of Bglll (manufactured by Takara Bio Inc.), and 2 L of 10 XH buffer were added to the solution to prepare a 20 L reaction solution. The extinguishing reaction was carried out at ° C for 16 hours.
  • pKANTEX93 (W097 / 10354) is dissolved in 17 ⁇ L of water, and 7.5 units of EcoRI and 2 ⁇ L of 10 XH buffer are added to the solution to prepare 20 ⁇ L of reaction solution.
  • the digestion reaction was performed at 37 ° C for 16 hours.
  • phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17 L of water.
  • 7.5 units of BamHI and 2 L of 10 XK buffer were added to the solution to prepare a 20 ⁇ L reaction solution, followed by digestion reaction at 37 ° C for 16 hours.
  • HP-1 haptoglobin DNA fragment (EcoRI-Bglll) and pKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each of about 1060 bp and 3 kbp.
  • the DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequence Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Applied) The base sequence was analyzed using Biosystems). As a result, plasmid pKAN-HP1 containing the cDNA sequence of HP-1 type haptoglobin was obtained (FIG. 3).
  • the plasmid pKAN-HPl prepared in the previous section 1 was introduced into FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)). These genes were introduced by the following procedure according to a known electoral position method [Cytotechnology, 3, 133 (1990)].
  • the reaction was linearized by digestion with C for 16 hours. After the reaction, the reaction solution is subjected to phenol / chloroform extraction treatment and Purification was performed by ethanol precipitation, and the linear plasmid was recovered.
  • FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137mmol / L KC1, 2.7mmol / L NaCl, 8.lmmol / L 8 x 10 7 cells suspended in Na HPO, 1.5mmol / L KH PO, 4.0mmol / L MgCl)
  • IMDM medium Life Technologies
  • Ushi fetal serum Life Technologies
  • 50 ⁇ g / mL gentamicin Nacalai Testa
  • the suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Subsequently, the medium was replaced with IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 200 nM MTX, and cultured again every 3-4 days for 18 days. Obtained MTX resistant strain HP1KO
  • HP1KO is suspended in IMDM medium supplemented with 5 mL of 10% urine fetal dialyzed serum, 50 ⁇ g / mL gentamicin and 200 nM MTX at a concentration of 3.0 ⁇ 10 5 cells / mL, and seeded in a T25 flask. Then, static culture was performed for 3 days. The culture supernatant after 3 days of culture was collected, and the amount of haptoglobin contained in the supernatant was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). It was confirmed that butoglobin was expressed.
  • Haptoglobin Kit manufactured by Tridelta Development Limited
  • the HP1KO strain is the name of HP1KO, and the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center, February 17, 2005 (Tsukuba Ito, Ibaraki Pref. Has been deposited as FERMBP-10249.
  • the culture supernatant of the HP1KO strain prepared in this way is then used for general purification of haptoglobin (development of follow-up medicines HP-1 type haptoglobin obtained by purifying using the 20th store, page 215-219) has fucose produced by the normal CHO / DG44 strain and has a sugar chain! Increased blood half-life was observed compared to HP-1 haptoglobin.
  • a FUT8 gene double knockout cell line producing human HP-2 haptoglobin was prepared by the method described below.
  • the solution after the PCR reaction was subjected to 2% agarose gel electrophoresis, and an approximately 1244 bp PCR amplified fragment (containing the HP-2 haptoglobin cDNA sequence) was purified using QIAquick Gel Extraction kit (manufactured by QIAGEN). After the obtained purified DNA fragment was dissolved in 17 L of water, 7.5 units of restriction enzyme EcoRI (Takara Bio) and 5 units of Bglll (Takara Bio), 2 L of 10 XH buffer were added to the solution. A 20 L reaction solution was prepared and the quenching reaction was carried out at 37 ° C for 16 hours.
  • pKANTEX93 (described in W097 / 10354) is dissolved in 17 ⁇ L of water, and 7.5 units of EcoRI and 2 ⁇ L of 10 XH buffer are added to the solution to prepare a 20 ⁇ L reaction solution. After the preparation, digestion reaction was performed at 37 ° C for 16 hours. After the reaction, phenol / chloroform extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17 L of water. Furthermore, 7.5 units of BamHI and 2 ⁇ L of 10 ⁇ K buffer were added to the solution to prepare a 20 ⁇ L reaction solution, followed by digestion reaction at 37 ° C. for 16 hours.
  • HP-2 haptoglobin DNA fragment (EcoRI-Bglll) and pKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (W / V) agarose gel electrophoresis, and each about 1240 bp A 3 kbp DNA fragment was purified using QIAquick Gel Extraction Kit (manufactured by QIAGEN).
  • the ligation reaction was performed for 16 hours.
  • the resulting plasmid DNA was used to transform Escherichia coli DH5 strain (manufactured by Toyobo Co., Ltd.) by the heat shock method.
  • the base sequence was analyzed using Biosystems).
  • plasmid pKAN-HP2 containing the cDNA sequence of HP-2 haptoglobin was obtained (Fig. 4).
  • the plasmid pKAN-HPl prepared in Example 1 was introduced into double knockout cells. This
  • FUT8 gene double knockout cells described in the literature (Biotechnology and Bioengineering 87, 614 (2004)) were mixed with K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L 8 x 10 7 cells suspended in Na HPO, 1.5mmol / L KH PO, 4.0mmol / L MgCl)
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, the medium exchange operation using IMDM medium supplemented with 10% urine fetal dialyzed serum, g / m Lgentamicin and 200 nM MTX was similarly repeated every 3-4 days for 18 days, and 200 nM MTX The resistant strain HP2KO was obtained.
  • MTX methotrexate
  • the resulting HP2KO was suspended in IMDM medium supplemented with 5 mL of 10% urine fetal dialyzed serum, 50 ⁇ g / mL gentamicin and 200 nM MTX at a concentration of 3.0 X 10 5 cells / mL, and T25
  • the flask was inoculated and statically cultured for 3 days.
  • the culture supernatant after 3 days of culture was collected, and the amount of haptoglobin contained in the supernatant was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited). It was confirmed that butoglobin was expressed.
  • HP2KO strain is the name of HP2KO, and the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on February 17, 2005 (Tsukuba Ibaraki 1-chome, 1st, 1st, 1st, 6th) Has been deposited as FERMBP-10250.
  • IMDM medium manufactured by Invitrogen
  • IMDM medium 1-fold concentration of IMDM medium (manufactured by Invitrogen)
  • PBS Dulbecco's PBS
  • the were detached cells were recovered by centrifugation operation performed by the conventional cell culture, 1 X 10 5 cells / mL IMDM-F BS (10 ) such that the density of - HT (1) medium was ⁇ Ka ⁇ After suspension, MNNG (manufactured by Sigma), which was not added or 0.1 g / mL alkylating agent, was added. 3 days at 37 ° C in CO incubator (TABAI)
  • the CHO / DG44 cell line was obtained.
  • GDP-mannose 4, 6- an enzyme that catalyzes the dehydration reaction of converting GDP-mannose into GDP-4-keto, 6-deoxy- GDP-mannose in each lectin-resistant CHO / DG44 cell line obtained in the previous section
  • the expression level of dehydratase was calculated using the RT-PCR method as follows.
  • RNA was prepared according to the instructions for use. Subsequently, using a SUPER SCRIPT First-Strand synthesis system for RT-PCR (manufactured by Invitrogen), single-stranded cDNA was synthesized from 5 g of each RNA in a 20 L reaction solution according to the attached instruction manual.
  • the expression level of the GDP-mannose 4,6-dehydratase gene in each lectin resistant CHO / DG44 cell line obtained in (1) of this section was analyzed.
  • the CHO cell-derived ⁇ -actin cDNA sequence (NCBI accession number: U20114) has a salted salmon sequence represented by SEQ ID NO: 22
  • a 24-mer synthetic oligo DNA primer having a 24 mol synthetic oligo DNA primer having a salt-and-sugar sequence represented by SEQ ID NO: 23 was prepared.
  • reaction solution containing 0.5 ⁇ L of single-stranded cDNA derived from each cell line prepared in this section (1) as a cage [1 X EX Taq Buffer (Takara Shuzo), 0.2 mM dNTP Prepare a mixture of 0.5 units of Ex Taq polymerase (Takara Shuzo), 0.5 ⁇ of synthetic DNA primers of SEQ ID NOS: 24 and 25], and use a DNA thermal cycler 480 (Perkin Elma) to make 94 ° After heating at C for 5 minutes, 30 cycles of 94 ° C for 1 minute and 68 ° C for 2 minutes were performed.
  • DNA thermal cycler 480 Perkin Elma
  • the CHO SM strain is a lectin that recognizes the same sugar chain structure as that recognized by LCA, that is, the N-glycoside-linked sugar chain reducing terminal. It was also resistant to other lectins that recognize sugar chain structures in which the N-acetylyldarcosamine residue at position 6 and fucose at position 1 are attached by an a bond.
  • a medium supplemented with endumerme lectin (Pisumsativum Agglutinin; hereinafter referred to as PSA, manufactured by Vector) with a final concentration of 1 mg / mL, or a white bamboo lectin with a final concentration of 1 mg / mL (Aleuria) aurantia Lectin; hereinafter referred to as AAL, manufactured by Vector, Inc.) was also resistant.
  • CHO / DG44 cells and the CHO SM strain obtained in the previous section were cultured using IMDM-FBS (10) -HT (1) medium in a T75 flask for adherent cell culture (manufactured by Grainer) until just before reaching confluence. Later, genomic DNA was prepared according to the method described in the literature [Nuccleic Acid Research, 3, 2303, (1976)], and the obtained genomic DNA was added to TE-RNase buffer (pH 8.0) [ 10 mmol / l Tris—HC1, lmmol / 1 EDTA, 200 ⁇ g / ml RNase A] 30 0 1 was dissolved overnight.
  • TE-RNase buffer pH 8.0
  • Genomic DNA was transferred to the membrane. After the transfer, the nylon membrane was heat treated at 80 ° C for 2 hours. Next, for the purpose of confirming the quality of the genomic DNA transferred to the nylon membrane, it is considered to exist evenly in the genome regardless of the cell line.
  • a probe for detecting the FU T8 gene was prepared as follows. First, 10 ⁇ g of plasmid m!
  • FUT8-pCR2.1 containing mouse FUT8 cDNA described in Example 11 of WO02 / 31140 was dissolved in 50 ⁇ l of M buffer (Takara Shuzo), and restriction enzyme Hindlll (Takara Shuzo) was dissolved. After overnight digestion, the reaction solution was replaced with H buffer (Takara Shuzo), and the digestion reaction was further performed overnight with the restriction enzyme EcoRI (Takara Shuzo). After completion of the reaction, the reaction solution was subjected to 2% agarose electrophoresis, and a 156 bp EcoRI-Hindlll fragment containing FUT8 gene exon 2 was purified.
  • the obtained DNA fragment (25 ng) was radiolabeled using [a- 32 P] dCTP 1.75 MBq and Megaprime DNA labeling system, dCTP (Amersham Biosciences).
  • hybridization was performed as follows. First, the above nylon membrane is sealed in a roller bottle, and 15 mL of a hybridization solution [4 X SSPE, 5 X Denhaldt, s solution, 0.5% (w / v) SDS, 0.1 mg / mL salmon sperm DNA] A prehybridization was performed at 65 ° C for 3 hours. Next, the 32 P-labeled probe DNA was heat denatured, put into a bottle, and heated at 65 ° C.
  • the nylon membrane was immersed in 50 mL of 2 X SSC-0.1% (w / v) SDS and heated at 65 ° C. for 15 minutes. After the above washing operation was repeated twice, the membrane was immersed in 50 mL of 0.2 X SSC-0.1% (w / v) S DS and heated at 65 ° C for 15 minutes. After washing, the nylon membrane was exposed to X-ray film at -80 ° C and developed. After development, the nylon membrane was boiled in a stripping solution [1% SDS, 0.1 X SSC] to peel off the probe and again subjected to hybridization with a different probe.
  • a stripping solution [1% SDS, 0.1 X SSC]
  • a probe specific for GMD gene exon 5 was prepared as follows. First, oligo DNA primers (SEQ ID NO: 26 and SEQ ID NO: 27) that specifically bind to exon 5 were designed based on the publicly known human GMD genomic DNA sequence (NCBI accession number: NT_034880). This region corresponds to nucleotide numbers 349 to 538 of the human GMD cDNA sequence shown in SEQ ID NO: 7.
  • a reaction solution of 100 ⁇ L containing lOng of plasmid pAGE2 49GMD described in Example 15 of WO02 / 31140 [ExTaq buffer (Takara Shuzo), 0.2 mmol / L dNTPs, 2.5 / z mol / L above gene-specific Primers (SEQ ID NO: 26 and SEQ ID NO: 27)] were prepared and subjected to polymerase chain reaction (PCR). PCR was performed under the conditions of 30 cycles, with a reaction consisting of 94 ° C for 5 minutes, 94 ° C for 1 minute, 58 ° C for 2 minutes, and 72 ° C for 3 minutes.
  • the reaction solution was subjected to 2% agarose electrophoresis, and an approximately 200 bp DNA fragment was purified.
  • the obtained DNA fragment 25ng, [ «- 32 P] dCTP 1.75MBq and Megapr ime DNA labeling system, and radiation-labeled using dCTP (manufactured by Amersham Biosciences).
  • the probe was hybridized to the nylon membrane shown above.
  • a specific fragment of GMD gene exon 5 was found in genomic DNA derived from CHO / DG44 cells, whereas a specific fragment of GMD gene exon 5 was found in genomic DNA derived from CHOSM strain! Was not detected at all. From the above results, it was shown that the CHO SM strain is a GMD knockout cell lacking at least the region containing exon 5 in the genomic region encoding GMD.
  • GMD knockout cell line producing human HP-1 haptoglobin is prepared by the following method
  • plasmid pKAN-HP1 prepared in Example 1 was introduced into the CHO SM strain prepared in Example 3. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)].
  • plasmid pKAN-H Prepare a reaction solution of 30 ⁇ g PI (20 ⁇ L NE Buffer 3 (New England Biolabs) and 200 ⁇ L 200 ⁇ L restriction enzyme Mlul (New England Biolabs) at 37 ° C for 16 hours. A linear reaction was achieved by performing a digestion reaction. After the reaction, the reaction mixture was purified by phenol / chloroform extraction treatment and ethanol precipitation to recover the linear plasmid.
  • Example 3 the CHO SM strain obtained in Example 3 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L). 8 x 10 7 cells suspended in MgCl)
  • IMDM medium Life Technologies
  • Ushi fetal serum Life Technologies
  • 50 ⁇ g / mL gentamicin Nacalai Testa
  • the suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium replacement using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 20 OnM MTX in the same manner every 3-4 days, and culture for 18 days. A resistant strain was obtained.
  • MTX methotrexate
  • the amount of haptoglobin contained in the culture supernatant of the 200 nM MTX resistant strain was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited).
  • the culture supernatant of the 200nM MTX-resistant strain produced in this way was obtained by purifying using the general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten No. 20, pages 215-219).
  • HP-1 type haptoglobin has an increased blood half-life compared to HP-1 type haptoglobin, which has a sugar chain to which fucose is bound, produced by the normal CHO / DG44 strain.
  • GMD knockout cell line producing human HP-2 haptoglobin is prepared by the following method
  • the plasmid pKAN-HP2 prepared in Example 2 was introduced into the CHO SM strain prepared in Example 3. These gene introductions were carried out by the following procedure according to a known electopore position method [Cytotechnology, 3, 133 (1990)].
  • Example 3 the CHO SM strain obtained in Example 3 was added to K-PBS buffer (137 mmol / L KC1, 2.7 mmol / L NaCl, 8. lmmol / L Na HPO, 1.5 mmol / L KH PO, 4.0 mmol / L). 8 x 10 7 cells suspended in MgCl)
  • IMDM medium Life Technologies
  • Ushi fetal serum Life Technologies
  • 50 ⁇ g / mL gentamicin Nacalai Testa
  • the suspension was suspended in 10 mL, and seeded in an adherent cell culture T 75 flask (manufactured by Grainer). The culture was performed under conditions of 5% CO and 37 ° C.
  • IMDM medium 10 mL of IMDM medium supplemented with analysis serum, 50 ⁇ g / mL gentamicin and 50 nM methotrexate (MTX) (manufactured by Sigma) was added. The culture was performed for 9 days while repeating this medium exchange operation every 3 to 4 days. Next, repeat the medium replacement using IMDM medium supplemented with 10% urine fetal dialyzed serum, 50 g / mL gentamicin and 200 nM MTX every 3-4 days. After culturing for 18 days, a 200 nM MTX resistant strain was obtained.
  • MTX methotrexate
  • the amount of haptoglobin contained in the culture supernatant of the 200 nM MTX resistant strain was measured using a Haptoglobin Kit (manufactured by Tridelta Development Limited).
  • the culture supernatant of the 200nM MTX-resistant strain produced in this way was obtained by purifying using the general purification method for haptoglobin (development of follow-on pharmaceuticals, Yodogawa Shoten No. 20, pages 215-219).
  • HP-2 haptoglobin was found to have an increased blood half-life compared to HP-2 haptoglobin, which has a sugar chain to which fucose is bound, produced by the normal CHO / DG44 strain.
  • yeasts Many types of yeast are known, but typical yeasts often used as hosts for expressing recombinant proteins include yeasts of the genera Pichia and Saccaromyces. . Normally, the main structure of N-linked sugar chains added to recombinant proteins expressed by these yeasts has a 2-residue N-acetyl darcosamine in the core part on the reducing end, and the non-reducing end side. It is known that this is a mannose-type sugar chain having 9 to several tens of mannose residues and several to several tens of mannose 6-phosphate residues at the branch of 1191 (2002)). Further, a high mannose type sugar chain having such a structure is also called a hypermannose type sugar chain.
  • the hybrid sugar chain which is an intermediate structure between the high-mannose sugar chain and the complex sugar chain, is mainly used as the structure of the N-linked sugar chain to be added.
  • the methods for preparing Pichia yeast strains and Saccharomyces yeast strains that express the HP-1 type or HP-2 type haptoglobin that have been carotenized are described below.
  • Pichia yeast strains that have disrupted the PN01 enzyme gene present on the genome Pichia yeast strains such as Pichia pastoris GTS115 (manufactured by Invitrogen Corp.) are used as genomic DNA, and PCR is used to perform PNOKphosphomannosylationof Pichia yeast.
  • N-linked oligosaccharides 1 Amplify the entire translation region of the gene (GenBank accession number: AB099514).
  • the amplified PN01 gene sequence with a length of about 3200 bases was replaced with the yeast orotidine-5'-phosphate decarboxylase (UR A3) gene (GenBank accession number: AF321098).
  • a plasmid for PN01 gene disruption is prepared by inserting into a vector such as PO vector (Invitrogen).
  • a vector such as PO vector (Invitrogen).
  • 100 g of this plasmid is linearized with a restriction enzyme, and then the gene is stably introduced into a Pichia yeast such as GTS115 strain, for example, by the electoral position method described in PichiaExpressionKit (manufactured by Invitrogen).
  • the introduced yeast is cultured at room temperature using YPD medium (Invitrogen) deficient in uracil, and genomic DNA is extracted from each of the grown colonies.
  • a yeast clone in which the PN01 locus is disrupted by homologous recombination is selected by amplifying the yeast PN01 locus sequence by PCR using this genomic DNA as a saddle type.
  • the structure of the main N-linked sugar chain expressed in Pichia yeast has 9 residues on the non-reducing end side, with 2 residues of N-acetylyldarcosamine in the core part on the reducing end side.
  • Pichia yeast strains such as Pichia pastoris X-33 (manufactured by Invitrogen), are used in a vertical form, and by PCR, Pichia yeast ⁇ -1,6-mannose transferase (OCH1) gene (GenBank accession) Number: AF540063) is amplified. Amplified about 2 800 salt
  • the OCH1 gene sequence of the base length was replaced with the yeast's orotidine-5'-phosphate decarboxylase (URA3) gene (GenBank accession number: AF3210 98) after replacing the 5 'terminal half sequence with pCR2.1 -A vector for disrupting the OCH1 gene is prepared by inserting into a vector such as TOPO vector (Invitrogen). Next, 100 g of this vector was linearly digested with the restriction enzyme Sfil (manufactured by New England Biolabs), and then the yeast yeast strain, for example, the above-mentioned item was obtained by the electoral position method described in Pichia Expression Kit (manufactured by Invitrogen).
  • Sfil restriction enzyme
  • Stable gene transfer is carried out to the PN01 gene disruption strain described in 1) or the Pichia pastoris JC308 strain.
  • the transfected yeast was cultured at room temperature in YPD medium (Invitrogen) lacking uracil and grown. Colony strength also extracts genomic DNA.
  • a yeast clonal strain in which the OCH1 locus is destroyed by homologous recombination is selected by amplifying the yeast OCH1 locus sequence by PCR using this genomic DNA as a saddle type.
  • the structure of the main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyl darcosamine in the core part on the reducing end side, and 8 structures on the non-reducing end side. It can be modified to a Man8 type high mannose type sugar chain having a structure in which a mannose residue is bound.
  • the cDNA encoding the active domain of is specifically amplified.
  • the amplified cDNA is ligated to the 5 'end of the cDNA sequence encoding the yeast ⁇ -mannosidase (MNS1) gene (GenBank accession number: M63598) leader peptide, and then the yeast expression vector pPICZ. (Invitrogen) and other vectors are inserted into the yeast endoplasmic reticulum for expression of -1,2-mannosidase.
  • this vector is stably introduced into the Pichia yeast strain in which both the PN01 gene and the OCH1 gene described in the previous section have been disrupted by homologous recombination by the electopore method.
  • the yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeosin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies.
  • a yeast clonal strain in which expression of the recombinant chimeric ⁇ -1,2-mannosidase is observed is selected by PCR using the first-strand cDNA prepared with this total RNA strength as a saddle type.
  • the structure of the main ⁇ -linked glycan expressed in Pichia yeast has 2 residues ⁇ -acetildarcosamine in the core part on the reducing end side and 5 on the non-reducing end side. It can be modified to a Man5 type high mannose type sugar chain having a structure in which the mannose residues are bound.
  • RNA Extract total RNA from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then prepare cDNA using the Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle type, and PCR is performed using specific primers and KOD polymerase (Toyobo) to encode the entire translation region of the yeast UDP-N-acetylyldarcosamine transporter. cDNA (Gen Bank accession number: AF106080) is specifically amplified.
  • the amplified cDNA of about 3700 bases in length is used as a restriction enzyme EcoRI cleavage site located downstream of the alcohol oxygenase promoter sequence of vectors such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen).
  • vectors such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen).
  • a vector that inserts between I cleavage sites and expresses UDP-N-acetyltilcosamine transporter in the Golgi apparatus of yeast is prepared.
  • this vector is stably introduced into the Pichia yeast strain introduced with the ⁇ -1,2-mannosidase gene described in the previous section by the electopore method.
  • the yeast after the gene introduction is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Nacalai Testa), and total RNA is extracted from each of the grown colonies.
  • a yeast clonal strain in which the expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
  • N-Acetyldarcosaminyltransferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a cage and using specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621). The amplified cDNA is linked to the 5 'end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752), and then expressed for yeast.
  • MNN9 yeast mannose transferase
  • vector pAUR123 manufactured by Tacarano
  • N-acetylyldarcosamine transferase- is inserted into the yeast Golgi.
  • a vector for expressing 1 is prepared.
  • this vector One is introduced into the Pichia yeast strain into which the UDP-N-acetylcyldarcosamine transporter gene described in the previous section has been introduced, by the lithium acetate method described in the manual attached to the expression vector pAUR123.
  • the yeast after gene transfer is cultured at room temperature in a YPD medium containing the drug mouthful brassin A (manufactured by Takara noisyo), and total RNA is extracted from each of the grown colonies.
  • a yeast clonal strain in which expression of recombinant N-acetylyldarcosamine transferase-1 is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
  • the structure of the main N-linked sugar chain expressed in Pichia yeast has 2 residues of N-acetyldarcosamine in the core at the reducing end and 5 at the non-reducing end. It can be modified to a noblebrid sugar chain with a structure in which one N-acetylyldarcosamine residue is added to the non-reducing end of the Man5 type high mannose sugar chain to which the mannose residue is attached. .
  • Pichia yeast strain that mainly expresses a hybrid-type sugar chain, which is an intermediate structure between a no-, immannose-type sugar chain and a complex-type sugar chain, as an N-linked sugar chain has been described above.
  • yeasts of the genus Saccharomyces can be mentioned as yeasts that are often used as hosts for expressing recombinant proteins.
  • a method for producing a Saccharomyces yeast strain that mainly expresses N-linked sugar chains and hybrid sugar chains as follows is described.
  • a yeast clone in which the OCH1 locus is destroyed by homologous recombination is selected.
  • the obtained Saccharomyces yeast strain in which the OCH1 gene was disrupted was derived from haploid cells according to the method of Sherman et al. (Methods'In'Enzymology 1 194, 21 (1991)), and then ⁇ -1,3-mannose.
  • a diploid zygote is formed by mixing with haploid cells of the mutant yeast strain LB1-10B (University of California Yeast Genetic Stock Center) in which the transferase (MNN1) gene is disrupted and culturing under nitrogen-deficient conditions .
  • the obtained zygote is cultured at room temperature in YPD medium lacking uracil and leucine, and genomic DNA is extracted from each colony force that has grown.
  • this genomic DNA was used as a saddle-shaped PCR method to determine the sequence of the yeast OCH1 locus (GenBan k clone session number: AF540063) and MNN1 locus sequence (GenBank accession number: AF540063L23753) are amplified to select yeast clones in which both OCH1 locus and MNN1 locus are destroyed.
  • the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has 2 residues N-acetylyldarcosamine in the core part on the reducing end side and 8 on the non-reducing end side. It can be modified into a Man8-type high mannose-type sugar chain having a structure in which the mannose residue is bound.
  • RNA extract total RNA from mold (Aspergillus saitoi) using RNeasy Mini Kit (Qiagen), and then prepare cDNA using Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is converted into a saddle type, and PCR using a specific primer and KOD polymerase (Toyobo Co., Ltd.) is performed to obtain a cDNA encoding the full-length translation region of mold a-1,2-mannosidase (GenBank Accession picker 1: Amplify D49827) specifically.
  • a specific primer and KOD polymerase Toyobo Co., Ltd.
  • the amplified cDNA with a length of about 1500 bases has a yeast endoplasmic reticulum localization signal peptide (Embo Journal 7, 913 (1988)), that is, histidine-aspartate, at the 3 'end from which the stop codon was deleted.
  • yeast endoplasmic reticulum localization signal peptide Embo Journal 7, 913 (1988)
  • histidine-aspartate at the 3 'end from which the stop codon was deleted.
  • this vector was stably added to the Saccharomyces yeast strain in which the a -1,6-mannose transferase gene and the a -1,3-mannose transferase gene described in the previous section were disrupted by the electopore method. Introduce.
  • the yeast after gene transfer is cultured at room temperature in a YPD medium (Invitrogen) containing zeocin (Invitrogen) and lacking uracil, and total RNA is extracted from each of the grown colonies.
  • a recombinant chimeric type yeast clone strain in which the expression of X-1,2-mannosidase was observed was selected by PCR using this cDNA with the total RNA strength prepared as a saddle type.
  • the main N-linked sugar chain structure expressed by Saccharomyces yeast has 2 residues of N-acetyldarcosamine in the core of the reducing end, and the non-reducing end It can be modified into a Man5 type high mannose type sugar chain having a structure in which 5 mannose residues are bonded to the side.
  • RNA Extract total RNA from yeast (Kluyveromyces lactis) using the RNeasy Mini Kit (Qiagen), and then prepare cDNA using the Superscript TM first-strand cDNA synthesis kit (Invitrogen) using this RNA as a cage. To do. Next, this cDNA is used as a saddle type, and PCR is performed using specific primers and KOD polymerase (Toyobo) to encode the entire translation region of the yeast UDP-N-acetylyldarcosamine transporter.
  • cDNA GenBank
  • the amplified cDNA having a length of about 3700 bases is combined with a restriction enzyme EcoRI cleavage site located downstream of a single alcohol oxygenase promoter sequence such as the yeast expression vector pPIC3.5K (manufactured by Invitrogen).
  • a vector that inserts between Not I cleavage sites and expresses UDP-N-acetyltilcosamine transporter in the yeast Golgi is prepared.
  • this vector is stably introduced into the Saccharomyces yeast strain into which the a-1,2-mannosidase gene has been introduced as described in the previous section by the electopore method.
  • the yeast after the gene introduction is cultured at room temperature in a YPD medium containing the drug G418 (manufactured by Nacalai Testa), and total RNA is extracted from each of the grown colonies.
  • a yeast clonal strain in which the expression of the recombinant UDP-N-acetylyldarcosamine transporter is observed is selected by PCR using the cDNA prepared from this total RNA as a saddle type.
  • N-acetylylcosamine transferase-1 (GenBank accession number) was obtained by performing PCR using human liver cDNA (Clontech) in a saddle shape and specific primers and KOD polymerase (Toyobo). : Amplify specifically the cDNA encoding the active domain of M55621).
  • the amplified cDNA contains the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end.
  • the cDNA sequence to be loaded After ligation of the cDNA sequence to be loaded, it is inserted between the restriction enzyme Kpnl cleavage site and the Xba I cleavage site located downstream of the alcohol dehydrogenase promoter sequence of vectors such as the yeast expression vector pAUR123 (Tacarano).
  • vectors such as the yeast expression vector pAUR123 (Tacarano).
  • a vector that expresses N-acetyltilcosamine transferase-1 in the Golgi apparatus of yeast is prepared.
  • this vector was introduced into the Saccharomyces yeast strain introduced with the UDP-N-acetylyldarcosamine transporter gene described in the previous section by the lithium acetate method described in the manual attached to the expression vector pAUR123. To do.
  • the yeast after the gene introduction is cultured at room temperature in a YPD medium containing a drug mouthful brassin A (manufactured by Takara Bio Inc.), and total RNA is extracted from each grown mouthpiece.
  • a yeast clone strain in which the expression of recombinant N-acetyl dalcosamine transferase-1 has been observed is selected by PCR using this cDNA, which has also been prepared for total RNA, in a vertical form.
  • the structure of the main N-linked sugar chain expressed in Saccharomyces yeast has a 2-residue N-acetylyldarcosamine in the core portion on the reducing end side and 5 in the non-reducing end side. It can be modified to a nodule-type sugar chain in which one N-acetylyldarcosamine residue is added to the non-reducing end of the Man5 type high mannose type sugar chain having a structure in which one mannose residue is bound.
  • human liver cDNA (manufactured by Invitrogen) is used as a cage and Pyrobest DNA polymerase (manufactured by Takara Bio Inc.) is used as an amplification enzyme.
  • the cDNA encoding the full-length mature haptoglobin is specifically amplified by the PCR reaction.
  • the obtained HP-1 type or HP-2 type haptoglobin cDNA is used as a restriction enzyme located downstream of the alcohol oxygenase promoter sequence of a vector such as the yeast expression vector pPIC6a (manufactured by Invitrogen).
  • a vector PPIC6a / HP1 for expression and a vector pPIC6a / HP2 for secreting and expressing mature human HP-2 haptoglobin are prepared.
  • a Pichia yeast strain that mainly expresses a hybrid-type sugar chain as an N-linked sugar chain, or a Saccharomyces yeast that mainly expresses a hybrid-type sugar chain as an N-linked sugar chain described in Section 9 of this Example Introduced into the strain by the lithium acetate method.
  • the yeast after the gene introduction is cultured at room temperature in a YPD medium (Invitrogen) containing the drug blasticidin (Invitrogen) to obtain a blasticidin-resistant colony.
  • the blasticidin-resistant colonies are transplanted into liquid YPD medium (Invitrogen) and cultured at 30 ° C for 24 hours or longer.
  • the culture supernatant obtained after the culture is analyzed using Haptoglobinkit (manufactured by Tridelta Development Limited) using a human plasma-derived haptoglobin preparation (manufactured by Mitsubishi Wellpharma) as a standard product.
  • Haptoglobinkit manufactured by Tridelta Development Limited
  • a human plasma-derived haptoglobin preparation manufactured by Mitsubishi Wellpharma
  • the recombinant HP-1 and HP-2 haptoglobins which have a hybrid sugar chain that does not contain fucose as an N-linked sugar chain, are secreted into the yeast culture supernatant, using the method of Krystal et al. (Bloodgl , 71 (1986)).
  • the purified HP-1 type and HP-2 type haptoglobin proteins can be analyzed for sugar chain structure according to the method of Skibeli et al. (Bloody, 3626 (2001)).
  • an N-linked glycan a Pichia that mainly expresses an ibubrid glycan with one N-acetyl darcosamine residue added to the non-reducing end of the Man5 type high mannose glycan.
  • Recombinant human HP-1 type and HP-2 type mainly using hybrid sugar chains that do not contain fucose as N-linked sugar chains, using yeast strains or similarly modified Saccharomyces yeast strains as hosts It was stated that haptoglobin can be prepared.
  • a method for producing a yeast strain expressing a recombinant human HP-1 type and HP-2 type haptoglobin mainly having a double-chain type sugar chain is described below.
  • the vector is inserted downstream of the promoter sequence of the vector to produce a vector that expresses ⁇ - mannosidase ⁇ in the yeast Golgi apparatus.
  • this vector expresses recombinant human HP-1 type or HP-2 type haptoglobin mainly having an glycan-linked glycan and an hybrid glycan as described in paragraph 11 of this Example. Stable introduction into yeast strains.
  • the fermented mother after gene transfer selects clones using auxotrophy and drug resistance as indicators, and then confirms the expression of chimeric ⁇ -mannosidase II by RT-PCR.
  • N-acetylyldarcosaminyltransferase is obtained by performing PCR using a specific primer and Pyrobest DNA polymerase (Takara Bio) from a human tissue (eg, liver-derived cDNA dnvitrogen). -Specific amplification of cDNA encoding the active domain of II (GenBank accession number: U15128). The amplified cDNA has a yeast mannose transferase (MNN9) gene (GenBank accession number: L23752) at the 5 'end.
  • MNN9 yeast mannose transferase
  • a vector for expressing transferase- ⁇ is prepared.
  • this vector was transformed into a chimeric a mannosidase described in the preceding paragraph in a yeast strain expressing recombinant human HP-1 or ⁇ ⁇ ⁇ ⁇ -2 haptoglobin mainly having a hybrid sugar chain as a ⁇ -linked sugar chain. Introduce stably to yeast strains that have stably introduced II. For the yeast after gene transfer, clones were selected using auxotrophy and drug resistance as indicators, and then the expression of chimeric N-acetylcolcamine transferase- ⁇ was confirmed by RT-PCR.
  • the sugar chain structure has two residues of N-acetyl darcosamine in the core portion on the reducing end side, and is linked to the non-reducing end side in a structure where three mannose residues are bifurcated. It can be modified into a complex double-stranded sugar chain containing fucose, with one N-acetylyldarcosamine residue added to each non-reducing end.
  • Amplify specific cDNA The amplified cDNA is inserted downstream of the expression motor sequence of an expression vector for yeast to produce a vector that expresses UDP-galactose-4-epimerase in the yeast cytosol.
  • yeast strain expressing recombinant human HP-1 type or HP-2 type haptoglobin mainly having an immature complex double chain type sugar chain as an N-linked type sugar chain as described in the previous section. In contrast, it is introduced stably. After the gene is introduced, clones are selected using auxotrophy and drug resistance as indicators, and then the expression of UDP-galactose-4-epimelase is confirmed by RT-PCR.
  • the amplified cDNA is ligated to the 5 ′ end of the cDNA sequence encoding the leader peptide of the yeast mannose transferase (MNN9) gene (GenBank accession number: L2375 2), and then expressed for yeast.
  • a vector is inserted downstream of the promoter sequence to express
  • this vector is used to express recombinant human HP-1 type or HP-2 type haptoglobin mainly having an immature complex double-stranded sugar chain as an N-linked sugar chain, as described in the previous section. Stable introduction into a yeast strain that has stably introduced chimeric ⁇ 1,4 galactosyltransferase into the yeast strain.
  • chimera 8 Major ⁇ -linked saccharides of recombinant HP-1 or ⁇ -2 haptoglobin expressed by yeast strains stably incorporating 1,4 galactosyltransferase
  • the chain structure has two residues ⁇ -acetyl darcosamine in the core part on the reducing end side, and the two non-reducing end side has three mannose residues in two branched structures. It can be modified into a complex double-stranded sugar chain in which one non-reducing end is added with one ⁇ -acetyldarcosamine residue and one galactose residue.
  • Recombinant HP-1 type or ⁇ -2 type haptoglobin which mainly has a complex double-stranded sugar chain with no fucose residue on the reducing end side and galactose added on the non-reducing end side, prepared in the previous section
  • the yeast strain to be expressed is seeded in a liquid YPD medium (manufactured by Invitrogen) and 24 hours at 30 ° C.
  • N-linked sugar chains secreted into the yeast culture supernatant Recombinant HP-1 type or HP-2 type haptoglobin with a complex double-stranded sugar chain containing fucose can be purified according to the method of Krystal et al. (Bloodgl, 71 (1986)) It is.
  • the purified HP-1 type or HP-2 type haptoglobin protein can be analyzed for the sugar chain structure according to the method of Skibeli et al. (Blood £ g, 3626 (2001)).
  • composition comprising a recombinant haptoglobin molecule having an N-glycoside-linked complex type sugar chain, wherein the N-glycoside-linked complex type sugar chain is N- It is possible to provide a haptoglobin composition which is a sugar chain in which fucose is bound to acetylyldarcosamine!

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention vise à procurer une composition d’haptoglobine comprenant une molécule d’haptoglobine génétiquement modifiée ayant une chaîne de sucre complexe à liaison de type N-glycoside, la chaîne de sucre complexe à liaison de type N-glycoside étant une chaîne de sucre dans laquelle le fucose n’est pas lié à la N-acétylglucosamine à l’extrémité réductrice de la chaîne de sucre, ainsi que son utilisation.
PCT/JP2006/307374 2005-04-06 2006-04-06 Composition comprenant une haptoglobine genetiquement modifiee WO2006109695A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2005-109874 2005-04-06
JP2005109874 2005-04-06

Publications (1)

Publication Number Publication Date
WO2006109695A1 true WO2006109695A1 (fr) 2006-10-19

Family

ID=37086970

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2006/307374 WO2006109695A1 (fr) 2005-04-06 2006-04-06 Composition comprenant une haptoglobine genetiquement modifiee

Country Status (1)

Country Link
WO (1) WO2006109695A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010023910A1 (fr) * 2008-08-30 2010-03-04 独立行政法人産業技術総合研究所 Procédé de modification d'une structure de chaîne de sucre dans une plante, et plante produite par le procédé
JP2013143969A (ja) * 2013-04-30 2013-07-25 National Institute Of Advanced Industrial Science & Technology 植物における糖鎖構造の改変方法及びその植物体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031140A1 (fr) * 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cellules produisant des compositions d'anticorps
WO2003085107A1 (fr) * 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
JP2005058111A (ja) * 2003-08-14 2005-03-10 Univ Osaka 糖鎖修飾制御方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031140A1 (fr) * 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cellules produisant des compositions d'anticorps
WO2003085107A1 (fr) * 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
JP2005058111A (ja) * 2003-08-14 2005-03-10 Univ Osaka 糖鎖修飾制御方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YAMANE-OHNUKI N. ET AL.: "Establishment of FUT8 knockout Chinese hamster ovary cells: an ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity", BIOTECH. BIOENG., vol. 87, no. 5, 5 September 2004 (2004-09-05), pages 614 - 622, XP002984450 *
YANG F. ET AL.: "Identification and characterization of human haptoglobin cDNA", PROC. NATL. ACAD. SCI. USA, vol. 80, no. 19, October 1983 (1983-10-01), pages 5875 - 5879, XP009020251 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010023910A1 (fr) * 2008-08-30 2010-03-04 独立行政法人産業技術総合研究所 Procédé de modification d'une structure de chaîne de sucre dans une plante, et plante produite par le procédé
JP2013143969A (ja) * 2013-04-30 2013-07-25 National Institute Of Advanced Industrial Science & Technology 植物における糖鎖構造の改変方法及びその植物体

Similar Documents

Publication Publication Date Title
JP6523404B2 (ja) 抗体組成物を生産する細胞
JP4628679B2 (ja) Ｇｄｐ−フコースの輸送に関与する蛋白質の活性が低下または欠失した細胞
WO2003085119A1 (fr) Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
JP5662149B2 (ja) 遺伝子組換えプロテインｓ組成物
JP4263191B2 (ja) アンチトロンビンｉｉｉ組成物の製造方法
JPWO2005035741A1 (ja) ゲノムが改変された細胞
US7691810B2 (en) Method of producing recombinant antithrombin III composition
JPWO2005035740A1 (ja) 無血清馴化したゲノム改変細胞
WO2006109695A1 (fr) Composition comprenant une haptoglobine genetiquement modifiee
WO2006109698A1 (fr) Composition comprenant une erythropoietine genetiquement modifiee
WO2006109696A1 (fr) Composition comprenant une folliculo-stimuline genetiquement modifiee

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 06731322

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP