WO2005035741A1 - ゲノムが改変された細胞 - Google Patents
ゲノムが改変された細胞 Download PDFInfo
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- WO2005035741A1 WO2005035741A1 PCT/JP2004/015318 JP2004015318W WO2005035741A1 WO 2005035741 A1 WO2005035741 A1 WO 2005035741A1 JP 2004015318 W JP2004015318 W JP 2004015318W WO 2005035741 A1 WO2005035741 A1 WO 2005035741A1
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Classifications
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
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- A61P37/08—Antiallergic agents
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- the present invention relates to an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose, a glycoprotein such as a cell in which a genomic gene is knocked out and an antibody using the cell. And a method for producing the same. '
- glycoproteins such as antibodies which are considered to be applied as pharmaceuticals, are produced by using genetic recombination technology. That is, glycoproteins such as antibodies are produced using animal cells, for example, CH0 cells derived from Chinese eight-muscle ovary tissue as host cells.
- the sugar chain structure of a glycoprotein depends on the host cell that produces the glycoprotein. For this reason, at present, a sugar chain for having optimal pharmacological activity is not always added to a glycoprotein.
- the sugar chain structure of a glycoprotein is a sugar chain gene, that is, a gene that encodes a glycosyltransferase that synthesizes a sugar chain and a gene that encodes a glycolytic enzyme that degrades a sugar chain, and also serves as a sugar donor to the sugar chain. It is regulated by the expression of genes encoding proteins responsible for various functions such as biosynthesis of intracellular sugar nucleotides and transport to the Golgi apparatus. So far, by introducing or mutating a gene encoding an enzyme or protein involved in the modification of these sugar chains into a host cell, the sugar chain structure of the glycoprotein produced by the host cell is changed. The possibility of control is shown.
- enzyme gene involved in sugar chain modification By introducing a gene encoding an enzyme involved in sugar chain modification (hereinafter referred to as “enzyme gene involved in sugar chain modification”) into a production cell, the sugar chain structure of the produced glycoprotein is altered. Have been attempted. Specifically, 1) by introducing a gene encoding rat) 3-galactoside (2,6-sialyltransferase) into CH0 cells, a large amount of sialic acid is added to the non-reducing terminal of the sugar chain. [J. Biol.
- Mutants in which the activity of a gene encoding a protein or an enzyme involved in sugar chain modification is altered include, for example, GA (wheat-germ agglutinin from T. vulgaris), ConA (concanaval in A from C. ensi formis). )> RIC (toxin from R. communis), L-PHA (leukoagglut inin from P. vulgaris), LCA (1 ent i 1 agglut iniii from L. cul inar is), PSA (from P. sat ivum) It has been obtained as a strain showing resistance to lectins without Pea lect in) [Somatic cell and Molecular Genet., 12, 51, 1986].
- glycoprotein having an altered sugar chain structure is produced by using a mutant having an altered activity of a gene encoding an enzyme or a protein involved in sugar chain modification as a host cell.
- I have.
- an antibody having a high mannose-type sugar chain structure was produced using a mutant CHO cell strain deficient in N-acetyltyl glucosamine transferase I (GnTI) activity.
- GnTI N-acetyltyl glucosamine transferase I
- GDP is an element that catalyzes the dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose in the de novo synthesis pathway of intracellular sugar nucleotide GDP-fucose. It has been reported that an antibody with high ADCC activity can be produced by using a strain with reduced activity of -mannose 4,6-dehydratase as a host cell [W000 / 61739, Journal of Biochemistry, Chemical Chemistry ( J. Biol. Chem.), 278, 3466, 2003, Journal of Biologics' Chemistry (J. Biol.
- N-glycoside-linked complex sugars A strain that is resistant to lectins recognizing a sugar chain structure in which the 6-position of N-acetyltilcosamine at the chain reducing end and the 1-position of fucose are ⁇ -linked, such as AAL (Lectin from Aleuria aurantia) CHO-ML strain, LCA (L. cul i CHO-LCA strains or Lec 13 strains that are resistant to lentil agglutinin (naris-derived) are used as host cells.
- AAL Lectin from Aleuria aurantia
- LCA L. cul i CHO-LCA strains
- Lec 13 strains that are resistant to lentil agglutinin (naris-derived) are used as host cells.
- other strains with reduced activity of GDP-mannose 4,6-dehydratase include PSA (P.
- sat ivum a mouse leukemia-derived cell line BW5147. Origin of Pea lec t in) tolerance established the PL R 1. as a mutant strain 3 is known [journal O Breakfast Bas Iorojikaru Chemistry (J. Biol. C em.) , 255, 9900, 1980] .
- 'none of these strains are completely gene-deficient, so the sugar chain structure that causes antibodies to exhibit high ADCC activity, that is, N-glycoside-linked sugars N It is difficult to completely suppress the addition of fucose to samin.
- mutants of such PL R 1. 3 and Lecl3 strain is obtained by introducing a random mutation by mutagenic agent treatment, have properties suitable necessarily as a strain used in the pharmaceutical manufacturing It is hard to say that there is.
- the present invention provides a cell capable of controlling a sugar chain structure modified to a glycoprotein that is a production protein, that is, a cell capable of producing a glycoprotein having high pharmacological activity.
- a method for producing a glycoprotein using the method, and a glycoprotein produced by the method is also a cell capable of controlling a sugar chain structure modified to a glycoprotein that is a production protein.
- the present invention relates to the following (1) to (24).
- a cell whose genomic gene has been knocked out an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose.
- the cell according to the above (5) which is a cell selected based on an index that shows a higher survival rate than the cell before the genomic gene is knocked out when cultured in.
- a method for producing a glycoprotein composition comprising using the cell according to any one of (1) to (: 10).
- the cells according to any one of (1) to (10) above are cultured in a medium, a glycoprotein composition is produced and accumulated in the culture, and the glycoprotein composition is collected from the culture. And a step of purifying the glycoprotein composition.
- transgenic non-human animal described in (14) above wherein the transgenic non-human animal is an animal selected from the group consisting of a red sea lion, a sheep, a goat, a pig, a white lion, a mouse, a rat, a chicken, a monkey, and a gray heron.
- the transgenic non-human animal or plant described or a progeny thereof is an animal selected from the group consisting of a red sea lion, a sheep, a goat, a pig, a white lion, a mouse, a rat, a chicken, a monkey, and a gray heron.
- a method for producing a glycoprotein comprising the steps of obtaining a tissue or body fluid containing the protein, and collecting and purifying a target glycoprotein composition from the obtained tissue or body fluid.
- a method for producing a glycoprotein composition comprising the steps of: culturing cells in a culture medium, producing and accumulating a glycoprotein composition in the culture, and collecting and purifying the glycoprotein composition from the culture.
- (22) A glycoprotein composition produced by the method according to any one of (11) to (: 13) and (19) to (21).
- a medicament comprising the composition according to (22) or (23) as an active ingredient.
- a cell in which the genomic gene of the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose of the present invention is knocked out (hereinafter referred to as “cell of the present invention”).
- One example is cells in which the genomic gene has been modified so that the activity of the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose is deleted.
- the genome has been modified such that the activity of the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose is deleted.
- Introducing a mutation refers to modifying the base sequence such as deleting, substituting, inserting and Z- or adding a base sequence on the genome.
- genomic gene knockout Complete suppression of the expression or function of the genomic gene thus modified is referred to as genomic gene knockout.
- a specific example in which a genomic gene is knocked out is an example in which all or part of a target gene has been deleted from the genome.
- the gene encoding the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose, the ATG region, catalytic active site, promoter region, etc. Deletion of part or all of the corresponding genome from the chromosome, or deletion of all of these alleles.
- at least the genomic region of the fifth exon is deleted on the chromosome. And all the alleles are deleted.
- all the alleles on the genome of the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose are knocked out.
- Specific examples of enzymes that catalyze the dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose include GDP-mannose 4,6-dehydratase.
- the GDP-mannose 4,6-dehydratase includes a protein encoded by the DNA of the following), (b), (c), (d), (e) or (f), or (g): (H), (i), (j), (k), (1), (m), (n) or (o).
- amino acids consist of a deletion, substitution, insertion, and Z or an added amino acid sequence, and are GDP-mannose 4,6-dehydratases.
- amino acids consist of a deletion, substitution, insertion, and Z or an added amino acid sequence, and are GDP-mannose 4,6-dehydrazine.
- a protein with a monozyme activity is a protein with a monozyme activity
- (m) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4 and having GDP-mannose 4,6-dehydratase activity
- (n) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 5 and having GDP-mannose 4,6-dehydratase activity
- (0) a protein consisting of an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6 and having GDP-mannose 4,6-dehydrase activity
- the DNA encoding the amino acid sequence of GDP-mannose 4,6-dehydratase has the following sequence: It hybridizes with a DNA having the nucleotide sequence represented by No. 1, 2 or 3 and a DNA having the nucleotide sequence represented by SEQ ID No. 1, 2 or 3 under stringent conditions, and has GDP-mannose 4, 6- Examples include DNA encoding an amino acid sequence having dehydratase activity.
- DNA that hybridizes under stringent conditions for example, a DNA such as a DNA having the base sequence represented by SEQ ID NO: 1, 2, or 3, or a fragment thereof, as a probe, DNA obtained by using a hybridization method, a plaque hybridization method, a Southern plot hybridization method, or the like can be used. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M of sodium salt using a filter on which DNA derived from colonies or plaques was immobilized. Then, filter with a 0.1- to 2-fold concentration of SSC solution (the composition of the 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 toM sodium citrate) at 65 ° C.
- SSC solution the composition of the 1-fold concentration SSC solution consists of 150 mM sodium chloride and 15 toM sodium citrate
- hybridizable DNA include a 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, and more preferably 80% or more. % 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: 4, 5 or 6 has an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added, and has GDP-mannose 4,6-dehydratase.
- Proteins having activity are described in Molecular Cloning 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.
- the number of amino acids to be deleted, substituted, inserted and / or added is one or more, and the number thereof is not particularly limited.
- a protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 4, 5 or 6 and having a GDP-mannose 4,6-dehydratase activity is defined as BLAST [ J. Mol. Biol., 215, 403 (1990)] and FASTA [Methods in Enzymology, 183, 63 (1990)].
- any method can be used as long as the desired genome can be modified, but a genetic engineering method is preferable.
- a specific method As a specific method,
- the cell of the present invention is a lectin-resistant cell line that recognizes a sugar chain structure in which the N-glycoside-linked complex type sugar chain reducing terminal N-acetyldarcosamine at position 6 and fucose at position 1 are ⁇ -linked. Selection can be made by using the selection method.
- Lectin-resistant cells are cells whose growth is not inhibited even when an effective concentration of lectin is given.
- the effective concentration is equal to or higher than the concentration at which cells before the genomic gene is knocked out (hereinafter also referred to as parent strain) cannot grow normally, and preferably the same as the concentration at which the cells before the genomic gene is knocked out cannot grow. Concentration, more preferably 2-5 times, even more preferably 10 times, most preferably 20 times or more.
- the effective concentration of lectin that does not inhibit the growth may be appropriately determined depending on the cell line, but is usually 10 ig / ml to 10fflg / m, preferably 0.5 mg / ml to 2, Omg / ml. .
- a lectin that recognizes a sugar chain structure in which the 6-position of N-acetyltilcosamine at the reducing end of an N-glycoside-linked sugar chain and the 1-position of fucose is a lectin that can recognize the sugar chain structure Any lectin can be used. Specific examples include lentil lectin LCA (Lentil Agglutinin from Lens Culinaris) Enduame lectin PSA (Pea Lectin from Pi sum sati food), Broad bean lectin VFA (Agglutinin from Vicia faba) Hylochawan Takelectin AAL (Aleuria aurantia-derived Lectin).
- the cell of the present invention may be any cell as long as it can express a glycoprotein, and includes yeast, animal cells, insect cells, plant cells, and the like. Specific examples of these cells are described below.
- the cells described in. Specific examples of animal cells include Chinese Eight Muster Ovarian Group Tissue-derived CHO cells, rat myeloma cell line YB2 / 3HL.P2.G11.16Ag.20 cell, mouse myeloma cell line NS0 cell, mouse myeloma cell line SP2 / 0-Agl4 cell, Syrian hamster kidney tissue-derived BHK cell, Examples include eight hybridoma cells producing antibodies, human leukemia cell line Namalba cells, embryonic stem cells, fertilized egg cells, plant cells, and the like.
- the above-mentioned myeloma cells, octibridoma cells, host cells for producing humanized antibodies or human antibodies, and transgenic non-human animals producing human antibodies are used for the production of antibodies and the like.
- Examples include embryonic stem cells or fertilized egg cells used for production, and plant cells used for producing transgenic plants that produce humanized antibodies and human antibodies.
- the cell of the present invention includes a glycoprotein composition produced by a cell before the genomic gene is knocked out, for example, a cell having the ability to produce an antibody composition having higher antibody-dependent cytotoxic activity than an antibody composition. About.
- NS0 cells before the genomic gene is knocked out are not particularly limited.
- NS0 cells before the genomic gene is knocked out include bio / technology (BIO / TECHNOLOGY), 10, 169 (1992), Biotechnology 1.
- NS0 cells described in literatures such as Bioengineering (Bioteclmol. Bioeng,), 73, 261 (2001).
- NS0 cell lines (RCB0213) registered with the RIKEN Cell Development Bank, or sublines obtained by adapting these strains to various serum-free media.
- SP2 / 0-Agl4 cells before the genomic gene was knocked out include the journal 'Ob' Immunol. (J. Immunol.), 126, 317, (1981), and Nichiya (Nature), 276, 269, (1978), and SP2 / 0-Agl4 cells described in the literature such as Human Antibodies and Hybridomas, 3, 129, (1992).
- SP2 / 0-Agl4 cells registered with the ATCC (ATCC CRL-1581) or a substrain (ATCC CRL-1581.1) obtained by adapting these strains to various serum-free media can also be mentioned.
- CH0 cells derived from Chinese hamster ovary tissue before the genomic gene was knocked out include Journal of Experimental Medicine, 108, 945 (1958), Proc. Natl. Acad. Sci. USA, 60, 1275 (1968), Genetics , 55, 513 (1968), Chromosoma, 41, 129 (1973), Methods in Cell Science, 18, 115 (1996), Radiation Research, 148, 260 (1997), Proc. Natl. Acad. Sci. USA, 77. Natl. Acad. Sci. 60, 1275 (1968), Cell, 6, 121 (1975), Molecular Cell Genetics, Appendix I, II (p883-900). CHO cells.
- CH0-K1 strain ATCCCCL-61
- listening strain ATCC CRL-9096
- Pro-5 strain ATCC CRL-1781 registered with the ATCC and commercially available CHO-S strain (Lifetechnologies Cat. # 11619) or these strains
- CHO-S strain Lifetechnologies Cat. # 11619
- Sub-strains adapted to various serum-free media are also included.
- G11. 16Ag. 20 cells before the genomic gene was knocked out include Y3 / Agl. 2, 3 cells (a cell line established from ⁇ ( « ⁇ -1631) Specific examples thereof include YB2 / 3HL. P2 described in references such as J. Cell. Bio, 93> 576 (1982) and Methods Enzymol. 73B, 1 (1981).
- the enzyme relating to fucose modification is inactivated. Therefore, in a cell in which a gene encoding a glycoprotein is introduced into the cell of the present invention, the produced glycoprotein is not subjected to fucose addition, and as a result, a glycoprotein composition having high physiological activity can be produced. .
- Glycoprotein compositions having high physiological activity include glycoprotein compositions with improved affinity for receptors, glycoprotein compositions with improved half-life in blood, and altered tissue distribution after blood administration. Glycoprotein compositions, such as glycoprotein compositions having improved interaction with proteins necessary for pharmacological activity.
- the glycoprotein produced in the present invention may be any glycoprotein composition having a fucose bond in the sugar chain structure of the produced protein when produced in cells before the genomic gene is knocked out. Any glycoprotein composition is included. Specific examples include antibodies, erythropoietin, thrombopoietin, histological plasminogen activator, properokinase, thrompomodulin, antithrompine I II, protein, blood coagulation factor VI I, and blood coagulation.
- Factor VI II Blood clotting factor IX, Blood clotting factor X, Blood clotting factor XII, Gonadotropin, Thyroid stimulating hormone, Epidermal growth factor (EGF), Hepatocyte growth factor (HGF), Keratinocyte growth factor, Actipin, Bone morphogenetic factor, stem cell factor (SCF), interferon and interferon) 3, interleukin-2, interleukin-2, interleukin-6, interleukin-10, interleukin-11, soluble interleukin-4 receptor, tumor Necrosis factor a, Dnasel, galactosidase, darcosidase, darcosele Such as mouth oxidase and the like.
- glycoproteins that bind to glycoproteins are composed of two types of sugar chains (N-glycoside-linked sugar chains) that bind to asparagine and sugar chains (0-glycoside-linked sugar chains) that bind to serine, threonine, etc. It is roughly divided into two types. These are collectively referred to as glycoside-linked sugar chains.
- N-glycoside-linked sugar chains have various structures [Experimental Biological Chemistry 23-Glycoprotein sugar chain research method (JSPS), edited by Reiko Takahashi (1989)]. And a common core structure represented by the following structural formula (I). Structural formula (I)
- anal Structural formula (I) In structural formula (I), the terminal of the sugar chain that binds to asparagine is called the reducing terminal, and the opposite side is the non-reducing terminal.
- N-glycoside-linked sugar chains examples include high-mannose-type sugar chains in which only mannose is bonded to the non-reducing end of the core structure, and galactose-N-acetyldarcosamine (hereinafter referred to as Gal-GlcNAc) at the non-reducing end of the core structure.
- Gal-GlcNAc galactose-N-acetyldarcosamine
- Complex type hereinafter referred to as “complex”
- N-acetylgalactosamine is ⁇ -linked to the hydroxyl group of serine or threonine, and further, galactose, ⁇ -acetyldarcosamine, ⁇ -acetylgalactosamine, fucose, or sialic acid.
- a linked sugar chain, xylose is linked to the hydroxyl group of serine, and galactose is linked to a hydroxyl group of hydroxy lysine.
- a sugar chain in which xylose is j3-linked to the hydroxyl group of serine usually, a plurality of sugars are bonded at the 4-position of the xylose, and a linear polysaccharide composed of a disaccharide is bonded to the end of the bonded sugar.
- the substance having such a sugar chain structure include cartilage proteoglycan.
- the substance having a sugar chain structure in which galactose is bonded to the hydroxyl group of hydroxylysin / 3 are collagen and the like.
- the glycoprotein composition refers to a composition comprising a glycoprotein molecule having an N-daricoside-linked complex type sugar chain or a ⁇ -glycoside-linked sugar chain. Since there are many sugar chains that bind to glycoproteins and their sugar chain structures are diverse, there are many combinations of sugar chains in the sugar chains of glycoproteins.
- the glycoprotein composition produced using the cells of the present invention may be composed of glycoprotein molecules having a single sugar chain structure as long as the effects of the present invention can be obtained. However, it may be composed of a glycoprotein molecule in which a plurality of different sugar chain structures are bonded.
- the glycoprotein composition produced by the cell of the present invention is preferably a sugar chain having no fucose bond to N-acetylgalactosamine at the reducing end of an N-glycoside-linked complex type sugar chain, And a glycoprotein composition having a sugar chain having no fucose linkage to the non-reducing terminal of N-acetylgalactosamine of a 0-glycoside-linked sugar chain.
- N-glycoside-linked complex-type sugar chain The sugar chain without fucose binding to N-acetylgalactosamine at the reducing end, and the fucos of the 0-glycoside-linked sugar chain to the non-reducing end of N-acetylgalactosamine
- a glycoprotein composition having a sugar chain having no bond includes, among all glycoside-linked sugar chains that bind to the amino acid region included in the glycoprotein composition, of the N-glycoside-linked complex type sugar chain reducing terminal.
- a sugar chain in which fucose is not bound to the N-acetylgalactosamine non-reducing terminal of a sugar chain in which fucose is not bound to N-acetyldarcosamine and a 0-daricoside-linked sugar chain is 100% And protein compositions. .
- the glycoprotein composition having a sugar chain to which fucose is not bound refers to a case in which fucose is substantially undetectable in the sugar chain analysis described in 6 below. Substantially undetectable means that it is below the detection limit of the measurement.
- a transgenic non-human animal or plant in which the genomic gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto and 6-deoxy-GDP-mannose is knocked out.
- a glycoprotein composition produced using the progeny thereof may be composed of a glycoprotein molecule having a single sugar chain structure as long as the effects of the present invention can be obtained. May be composed of glycoprotein molecules having different sugar chain structures.
- glycoprotein composition preferably, all glycoside-linked sugar chains that bind to the amino acid region contained in the glycoprotein composition are N-glycidyl glucosamine at the N-glycoside-linked complex type sugar chain reducing terminal.
- glycoprotein compositions having a sugar chain to which no fucose is bonded at the non-reducing end of N-acetylgalactosamine of the 0-glycoside-linked sugar chain are described.
- a non-human animal or plant before its genomic gene is knocked down, or a progeny thereof (hereinafter referred to as a parent individual) produces a fucose-modified glycoprotein composition.
- Transgenic non-human animals or plants, or their progeny in which the genomic gene of an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose has been knocked out, It can be produced using the embryonic stem cell, fertilized egg cell, or plant cell of the present invention.
- the glycoprotein composition produced according to the present invention among all glycoside-linked sugar chains bound to the amino acid region to which the sugar chain is bound, fucose is added to N-acetylethylglucosamine at the N-glycoside-linked complex type sugar chain reducing terminal.
- the percentage of glycans that are not bound to the non-reducing end of NT-cetylgalactosamine non-reducing end of glycans that are not linked to the genomic gene is knocked out.
- the glycoprotein composition produced according to the invention has a higher biological activity than the glycoprotein composition produced by the cell or parent individual.
- a sugar chain in which fucose is not bound to N-acetyldarcosamine at the N-glycoside-linked complex type sugar chain reducing end which is contained in a composition comprising a glycoprotein molecule that binds a glycoside-linked sugar chain, and 0-glycoside
- the proportion of sugar chains in which fucose is not bound to the N-acetylgalactosamine non-reducing terminal of the linked sugar chain can be determined by a known method such as hydrazinolysis or enzymatic digestion from glycoprotein molecules.
- the antibody composition refers to a composition comprising an antibody molecule having an ⁇ -glycoside-linked complex type sugar chain in the Fc region.
- Antibodies are tetramers in which two types of polypeptide chains, heavy and light, are associated with each other by two molecules. About one-fourth of the N-terminal side of the heavy chain and about one-half of the N-terminal side of the light chain (each more than 100 amino acids) are called variable regions and are rich in diversity and directly involved in antigen binding I do. Most of the part other than the variable region is called the constant region. Antibody molecules are classified into IgG, IgM, IgA, IgD, and IgE classes according to the homology of the constant regions.
- the IgG class is further classified into IgG1 to IgG4 subclasses based on the homology of the constant regions.
- the heavy chain is divided into four immunoglobulin domains, VH, CH1, CH2, and CH3, from the N-terminal side.There is a highly mobile peptide region called the hinge region between CH1 and CH2, and CH1 and CH2 Separated.
- the structural unit consisting of CH2 and CH3 after the hinge region is called the Fc region, to which N-glycoside-linked sugar chains are bound. This region is where the Fc receptor and complement bind.
- the Fc region of the antibody molecule has a region where N-glycoside-linked sugar chains bind one by one, two sugar chains are bound per antibody molecule.
- the N-glucoside-linked sugar chain that binds to an antibody molecule has various structures, and any of the sugar chain structures includes the core structure represented by the structural formula (I).
- the antibody composition produced using the cells of the present invention may be composed of an antibody molecule having a single sugar chain structure as long as the effects of the present invention can be obtained, It may be composed of a plurality of antibody molecules having different sugar chain structures.
- the antibody composition produced by the cells of the present invention is preferably N-acetylethyl glucoside at the reducing end of the sugar chain among all glycoside-linked complex-type sugar chains binding to the Fc region contained in the antibody composition.
- An antibody composition having a sugar chain in which fucose is not bound to samin is exemplified.
- the cell of the present invention can produce an antibody composition having higher antibody-dependent cytotoxic activity than an antibody composition produced by a cell before the genomic gene is knocked out.
- N-glycoside-linked complex-type sugar chains can be mentioned.
- the antibody composition produced using the progeny thereof may be composed of an antibody molecule having a single sugar chain structure or a plurality of different antibody molecules as long as the effects of the present invention can be obtained. It may be composed of an antibody molecule having a sugar chain structure.
- fucose binds to N-acetyldarcosamine at the reducing end of the sugar chain among all glycoside-linked complex-type sugar chains that bind to the Fc region contained in the antibody composition.
- An antibody composition having an unreacted sugar chain is obtained.
- Antibody composition having a sugar chain in which fucos is not bound to N-acetyldarcosamine at the reducing end of the sugar chain among all glycoside-linked complex sugar chains that bind to the Fc region contained in the antibody composition
- An antibody composition having a sugar chain to which fucose is not bound refers to a case where fucose is substantially undetectable in the sugar chain analysis described in 6 below. Substantially undetectable means that it is below the detection limit of the measurement.
- Transgenic non-human animals or plants, or their progeny in which the genomic gene of the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GD-mannose has been knocked out, It can be produced using the embryonic stem cell, fertilized egg cell, or plant cell of the present invention.
- the sugars in which fucose is not bound to N-acetyltyldarcosamine at the reducing end of the sugar chain among all the N-daricoside-linked complex-type sugar chains binding to the Fc region If the ratio of the chains is higher than the antibody composition produced by the cell or parent individual from which the genomic gene is knocked out, the antibody composition obtained by the present invention is obtained from the antibody molecule produced by the cell or parent individual. A higher ADCC activity than the antibody composition. .
- ADCC activity means that in vivo, antibodies that bind to cell surface antigens, such as tumor cells, activate effecta cells through the binding of the antibody Fc region to the Fc receptor present on the cell surface.
- cell surface antigens such as tumor cells
- effector cells include killer cells, natural killer cells, activated macrophages, and the like.
- the ratio of sugar chains in which fucose is not bonded to N-azetylglucosamine at the reducing end of the sugar chains contained in the composition comprising an antibody molecule having an N-glycoside-linked complex type sugar chain in the Fc region is as follows: Using known methods such as hydrazinolysis and enzymatic digestion from molecules [Biochemical Experiment 23-Glycoprotein Glycoprotein Research Method (Gakkai Shuppan Center) ⁇ Reiko Hashihashi (1989) '], release sugar chains and release them.
- the sugar chain can be determined by fluorescent labeling or isotope labeling, and the labeled sugar chain is separated by a chromatography method.
- the antibody of the present invention includes an antibody that recognizes a tumor-associated antigen, an antibody that recognizes an antigen associated with allergy or inflammation, an antibody that recognizes an antigen associated with a circulatory disease, and an antigen that is associated with an autoimmune disease.
- the antibody is an antibody that recognizes an antigen, or an antibody that recognizes an antigen associated with viral or bacterial infection.
- the antibody class is preferably IgG.
- Antibodies that recognize tumor-associated antigens include anti-GD2 antibody (Anticancer Res., 13, 331, 1993), anti-GD3 antibody (Cancer Immunol.Immunoter., 36 ⁇ 260, 1993), and anti-GM2 antibody (Cancer Res. , 54, 1511, 1994), anti-HER2 antibody (Pro Natl. Acad. Sci. USA, 89, 4285, 1992), anti-CD52 antibody (Nature, 332> 323, 1988), anti-MAGE antibody (British J.
- Anti-PMSA antibody J. Urology, 160, 2396, 1998), anti-vascular endothelial cell growth factor Antibody (Cancer Res., 57, 4593, 1997) or anti-vascular endothelial growth factor receptor antibody (Oncogene, 19, 2138, 2000), anti-CA125 antibody, anti-17-1A antibody, anti-integrin ⁇ ) 33 antibody , Anti-CD33 antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DR antibody, anti-CD20 antibody, anti-CD19 antibody, anti-EGF receptor antibody (Immunology Today, 21, 03, 2000), anti-CD10 antibody (American Journal of Clinical Pathology, ⁇ 3, 374, 2000).
- Antibodies that recognize allergic or inflammation-related antigens include interleukin-6 antibody (Immunol. Rev., 121, 5, 1992) and anti-interleukin-6 receptor antibody (Molecular
- anti-interleukin-4 receptor antibody L Immunol. Met., 217, 41, 1998), anti-tumor necrosis factor antibody (Hybridoma, 13, 183, 1994), anti-tumor necrosis factor receptor antibody (Molecular Pharmacol) , 58, 237, 2000), anti-CCR4 antibody (Nature, Sato '776, 1999), anti-chemokine antibody (J. Immunol. Meth., 174, 249, 1994), anti-chemokine receptor antibody (J. Exp. Med., 186, 1373, 1997), anti-IgE antibody, anti-CD23 antibody, anti-CDlla antibody (Illegal Unolgy Today, 21, 403, 2000), anti-CRTH2 antibody (J. Immunol., 162, 1278, 1999) And anti-CCR8 antibody (W099 / 25734), anti-CCR3 antibody (US6207155) and the like.
- Antibodies that recognize antigens associated with cardiovascular diseases include anti-GpIIb / IIIa antibodies (J. Immunol., 152, 2968, 1994), anti-platelet-derived growth factor antibodies (Science, 253, 1129, 1991), anti-platelets Derived factor receptor antibody (J. Biol. Chem., 272, 17400, 1997) or anticoagulation factor antibody (Circulation, 101, 1158, 2000).
- Antibodies that recognize antigens associated with autoimmune diseases include anti-self DNA antibodies (Immunol. Letters, 72, 61, 2000), anti-CDlla antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin ⁇ 4 jS7 antibody, anti-CD40L antibody, anti-IL -2 receptor antibody (I band unol ogy Today, 21, 403, 2000).
- Antibodies that recognize antigens associated with viral or bacterial infection include anti-gp120 antibody (Struture, 8, 385, 2000), anti-CD4 antibody (J. Rheumatology, 25, 2065, 1998), and anti-CCR4 antibody And anti-verotoxin antibodies (J. Clin. Microbiol., 37, 396, 1999).
- the antibody molecule includes any molecule that contains the Fc region of the antibody. Specific examples include an antibody, an antibody fragment, and a fusion protein containing an Fc region.
- the antibody may be any protein that is produced in vivo by an immune reaction as a result of stimulating a foreign antigen and has an activity of specifically binding to the antigen.
- antibodies secreted by hybridoma cells produced from animal spleen cells and antibodies produced by genetic recombination technology that is, antibody expression vectors into which antibody genes have been inserted, are obtained by introducing them into host cells
- Antibodies and the like Specific examples include antibodies produced by hybridomas, humanized antibodies, human antibodies, and the like.
- the hybridoma has a desired antigen specificity obtained by cell fusion of B cells obtained by immunizing a non-human mammal with an antigen and myeloma cells derived from mice, rats, etc. mono Refers to cells that produce clonal antibodies.
- humanized antibody examples include a human chimeric antibody and a human CDR-grafted antibody.
- Human chimeric antibodies are composed of antibody heavy chain variable regions (hereinafter also referred to as HV or VH as V regions) and antibody light chain variable regions (hereinafter light chains are referred to as LV or VL as L chains) in non-human animals. ) And the heavy chain constant region of a human antibody (hereinafter also referred to as CH) and the light chain constant region of a human antibody (hereinafter also referred to as CL).
- HV or VH as V regions antibody heavy chain variable regions
- LV or VL antibody light chain variable regions
- CH heavy chain constant region of a human antibody
- CL light chain constant region of a human antibody
- any mouse, rat, hamster, rabbit and the like can be used as long as it can produce a hybridoma.
- Human-type chimeric antibodies are obtained from cDNAs encoding VH and VL from hybridomas that produce monoclonal antibodies, and converted into expression vectors for host cells that have genes encoding human human CH and human antibody CL.
- a human-type chimeric antibody expression vector can be constructed by inserting each of them, and can be expressed and produced by introducing the vector into a host cell.
- any CH may be used as long as it belongs to human immunoglobulin (hereinafter, referred to as hlg), but the hlgG class is preferable, and hIgGl, h IgG2, Any of the subclasses hIgG3 and MgG4 can be used.
- the CL of the human chimeric antibody may be any CL as long as it belongs to hlg, and ⁇ class or ⁇ class CL can be used.
- the human CDR-grafted antibody refers to an antibody obtained by grafting the VH and VL CDR amino acid sequences of a non-human animal antibody to the appropriate positions of ⁇ and VL of a human antibody.
- the human CDR-grafted antibody is constructed by constructing a cDNA encoding the V region obtained by grafting the VH and VL CDR sequences of a non-human animal antibody to the VH and 1 VL CDR sequences of any human antibody.
- the human CDR-grafted antibody expression vector is constructed by inserting the expression vector into a host cell expression vector having a gene encoding the CH and the human antibody CL, and introducing the expression vector into the host cell.
- Type CDR-grafted antibodies can be expressed and produced.
- the CH of the human CDR-grafted antibody may be any CH as long as it belongs to Mg; however, the hlgG class is preferable, and any subclass such as hIgGl, hIgG2, hIgG3, and MgG4 belonging to the hlgG class should be used. Can be.
- the CL of the human CDR-grafted antibody may be of any type as long as it belongs to hlg, and may be of class or ⁇ class.
- Human antibody originally refers to an antibody that naturally exists in the human body, but human antibody phage libraries and human antibody production made by recent advances in genetic engineering, cytology, and developmental engineering technologies Antibodies obtained from transgenic animals or transgenic plants producing human antibodies are also included. '
- Antibodies present in the human body include, for example, isolating human peripheral blood lymphocytes and The antibody can be obtained by obtaining and culturing a lymphocyte that produces the antibody by purifying and immortalizing and cloning, and then purifying the antibody from the culture in the lymphocyte.
- the human antibody phage library 1 is a library in which antibody fragments such as Fab and single chain antibody are expressed on the phage surface by introducing an antibody gene prepared from human B cells into the phage gene. From the library, phages expressing antibody fragments having the desired antigen-binding activity can be recovered using the binding activity to the substrate on which the antigen is immobilized as an index. The antibody fragment can be further converted to a human antibody molecule consisting of two complete H chains and two complete L chains by genetic engineering techniques.
- a human antibody-producing transgenic non-human animal refers to an animal in which a human antibody gene has been integrated into cells.
- a human antibody-producing transgenic animal can be prepared by introducing a human antibody gene into mouse embryonic stem cells, transplanting the embryonic stem cells into an early embryo of another mouse, and then developing the embryo.
- a human antibody-producing transgenic animal can be produced by introducing a human antibody gene into a fertilized egg of an animal and generating the fertilized egg.
- the method for producing human antibodies from human antibody-producing transgenic animals is as follows: human antibody-producing hybridomas are obtained by the usual method for producing hybridomas in mammals other than humans, and cultured to obtain human antibodies. Can produce and accumulate human antibodies.
- transgenic non-human animals examples include porcupines, sheep, goats, pigs, pomas, mice, rats, birds, monkeys, and egrets.
- the antibody may be an antibody that recognizes a tumor-associated antigen, an antibody that recognizes an antigen that is associated with allergy or inflammation, an antibody that recognizes an antigen that is associated with cardiovascular disease, or an antigen that is associated with an autoimmune disease.
- the antibody is preferably an antibody that recognizes, or an antibody that recognizes an antigen associated with a virus or bacterial infection, and a human antibody whose antibody class is IgG is preferable.
- the antibody fragment refers to a fragment containing at least a part of the Fc region of the antibody.
- the Fc region refers to a region at the C-terminal side of the H chain of the antibody, a CH2 region and a CH3 region, and includes a natural type and a mutant type thereof.
- At least a part of the Fc region refers to a fragment preferably containing a CH2 region, more preferably a region containing the first aspartic acid present in the CH2 region.
- Specific examples of antibody fragments include H chain monomers and H chain dimers.
- the fusion protein having an Fc region may be any substance obtained by fusing an antibody or a fragment of the antibody containing the Fc region of the antibody with a protein such as an enzyme or cytokin (hereinafter, referred to as an Fc fusion protein). It can be anything.
- the cell of the present invention can be prepared by the method described below.
- the cells of the present invention are prepared by targeting the genomic gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose, and using a gene disruption method. be able to.
- Specific examples of enzymes that catalyze the dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose include GDP-mannose 4,6-dehydratase. It is possible.
- the method of gene disruption includes any method that can disrupt the gene of the target enzyme. Examples thereof include a homologous recombination method, an RNA-DNA oligonucleotide (RD D) method, a method using a retrovirus, and a method using a transposon. These will be described in detail below.
- RD D RNA-DNA oligonucleotide
- the cells of the present invention target the gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose, and homologous recombination of the target gene on the chromosome is performed. It can be produced by modification. .
- Modification of the target gene on the chromosome can be performed using the method described in Manipuling the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994) (hereinafter, ⁇ manipulating the mouse. Laboratory Manual), Gene Targeting, A
- a genome DNA of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose is prepared.
- a target gene to be modified based on the nucleotide sequence of the genomic DNA for example, a structural gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose, or Is a target vector for homologous recombination of the promoter gene.
- the cells of the present invention can be prepared by selecting cells that have undergone homologous recombination.
- Host cells include yeast, animal cells, insect cells, plant cells, and other genes containing enzymes that catalyze the dehydration reaction that converts the target GDP-mannose to GDP-4-keto, 6-dexoxy-GDP-mannose. Any of these can be used. Specific examples include the cells described in 3. below.
- Methods for obtaining cDNA and genomic DNA of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose include, for example, the methods described below. .
- a cDNA library is screened, and an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-dexoxy-GDP-mannose is coded. You can get cDN A.
- the mRNA of various host cells may be commercially available (eg, Clontech) or may be prepared from various host cells as follows. Methods for preparing total RNA from various host cells include guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], guanidine acid phenol / phenol and phenol ⁇ Black mouth alum (AGPC) method [Analytical Biochemistry (162), 156 (1987); Experimental Medicine, 9, 1937 (1991)].
- mRNA as a method for preparing mRNA as poly (A) + RNA from total RNA, an oligo (dT) -immobilized cellulose column method (Molecular Cloning, 2nd edition) and the like can be mentioned.
- mRNA can be prepared by using a kit such as Fast Track mRNA Isolation Kit (Invitrogen) and Quick Prep mRNA Purification Kit (Pharmacia).
- the cDNA library can be prepared by the method described in Molecular 'Cloning 2nd edition, Current Protocols-in-Molecular' biology, A Laboratory Manual, 2nd Ed. (1989), or a commercially available kit. E.g., Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) and the method using ZAP-cDNA Synthesis Kit (ST Tetsu GENE).
- any phage vector, plasmid vector, or the like can be used as long as it can replicate in E. coli K12 strain.
- ZAP Express [STRATAGENE, Strategies, 5, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)] , Lambda ZAP II (STRATAGENE), AgtlO, Agtll [DNAcloning, APractical Approach], ⁇ , 49 (1985)], ATriplEx (Clontech), AExCell (Pharmacia) ), T7T318U (Pharmacia), pcD2 [Molecular 'Cellular' Biology (Mol. Cell. Biol.), 3, 280 (1983)] and pUC18 [Gene, 33-103 (1985)], etc. Can be given.
- Escherichia coli As a host microorganism for preparing the cDNA library, any microorganism can be used, but Escherichia coli is preferably used. Specifically, Escherichia coli XL Blue Blue '[STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coH Y1088 [Science, 222, 778 (1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)], Escherichia 2 Hiring 522 [Journal 'ob' Molecular 'Piology (J.
- This cDNA library may be used as it is for subsequent analyses, but the oligocap method developed by Sugano et al. [Gene, in order to reduce the proportion of incomplete cDNA and to obtain full-length cDNA as efficiently as possible. (Gene), 138> 171 (1994); Gene, 200, 149 (1997); Protein nucleic acid enzyme, ⁇ , 603 (1996); Experimental medicine, ⁇ , 2491 (1993); cDNA cloning. (Yodosha) (1996); Method for Generating Gene Library (Yodosha) (1994) 3 A cDNA library prepared using (3) may be used for the following analysis.
- the 5 ′ of the nucleotide sequence predicted to encode the amino acid sequence PCR was performed using the generated cDNA library as a ⁇ type PCR primer (PCR Protocols, Academic Press ( 1990)] to obtain a gene fragment encoding an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto and 6-deoxy-GDP-mannose. it can.
- the obtained gene fragment is a DNA encoding an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose.
- Sanger et al.'S didoxy method [Procedings of the National Academy's Ob Science (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)] or the ABI. It can be confirmed by analysis using a base sequence analyzer such as PRISM377 DNA Sequencer (manufactured by PE Biosystems).
- colony hybridization or plaque hybridization (Molecular Cloning 2nd Edition) is performed on a pair of cDNA or cDNA libraries synthesized from mRNA contained in various host cells.
- plaque hybridization Molecular Cloning 2nd Edition
- DNA an enzyme that catalyzes the dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose.
- the primer used to obtain a gene fragment encoding an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose is included in various host cells.
- screening is performed using the PCR method to reduce the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose. It is also possible to obtain the DNA of the catalyzing enzyme.
- nucleotide sequence of DNA encoding the enzyme that catalyzes the dehydration reaction that converts the obtained GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose from the end
- a commonly used nucleotide sequence analysis method eg For example, the dideoxy method of Sanger et al. [Proceedings of the National Academic Symposium, Pro Natl. Acad. Sci. USA, 74, 5463 (1977)] or ABI
- the base sequence of the DNA is determined by analysis using a base sequence analyzer such as PRISM377 DNA Sequencer (manufactured by PE Biosystems).
- the nucleotide sequence of a gene encoding an enzyme that catalyzes a dehydration reaction that converts GDP-mannose obtained by the above method into GDP-4-keto, 6-deoxy-GDP-mannose is, for example, SEQ ID NO:
- the base sequence described in 1, 2, or 3 can be mentioned.
- the protein is chemically synthesized using a DNA synthesizer such as the DNA synthesizer model 392 manufactured by Parkin Pharma Inc. using the phosphoramidite method. It is also possible to obtain cDNA for an enzyme that catalyzes the dehydration reaction that converts -mannose to GDP-4-keto, 6-deoxy-GDP-mannose. Examples of a method for preparing genomic DNA of an enzyme that catalyzes a dehydration reaction for converting GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose include the following methods. '
- Examples of the method for preparing genomic DNA include known methods described in Molecular II Cloning, 2nd Edition, Current Protocols, "In” Molecular Biology, and the like. Also, by using a genome DNA library screening system (Genome Systems) or Universal GenomeWalker TM Kits (CL0NTECH), GDP-mannose is converted to GDP-4-keto and 6-doxy-GDP-mannose. The genomic DNA of the enzyme that catalyzes the dehydration reaction can be isolated.
- the target vector for homologous recombination of the target gene is Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), and the production of mutant mice using ES cells (Yodosha). ) And the like.
- the target vector can be either a rib racement type or an insertion type.
- the method for introducing a recombinant vector suitable for various cells described in 3. below can be used. .
- Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Production of mutant mice using ES cells (Yodosha) ), Positive selection, promoter selection, negative selection, poly A selection, etc.
- Methods for selecting the desired homologous recombinant from the selected cell lines include the Southern hybridization method for genomic DNA (Molecular Cloning, 2nd edition) and the PCR method (PCR protocol). (PCR Protocols), Academic Press (1990)] and the like.
- Homologous recombinants can also be obtained by using the change in enzyme activity that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose as an indicator.
- a specific method for example, there is a method for selecting a transformant described below.
- Biochemical methods include, for example, a method of evaluating enzyme activity using an enzyme-specific substrate.
- Examples of the genetic engineering method include Northern analysis for measuring the mRNA amount of an enzyme gene and RT-PCR method.
- a method of selecting cells based on knockout of a genomic gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose, resulting in a change in a trait resulting from the knockout examples include a method of selecting a transformant using the sugar chain structure of the produced antibody molecule as an index and a method of selecting a transformant using the sugar chain structure of a glycoprotein on the cell surface as an index.
- a method for selecting a transformant using the sugar chain structure of the production body molecule as an index the method described in 6. below can be mentioned.
- a method for selecting a transformant using the sugar chain structure of a glycoprotein on the cell surface as an index is as follows: 6-position of N-glycidylcosamine at the reducing end of N-glycoside-linked sugar chain and 1-position of fucose are a-linked.
- a method for selecting a strain that is resistant to a lectin that recognizes the sugar chain structure described above can be used. Specific examples thereof include a method using a lectin described in Somatic Cell Mol. Genet., 12, 51, (1986) and the like.
- any lectin can be used as long as the lectin recognizes a sugar chain structure in which the 6-position of N-acetyldarcosamine at the reducing end of the N-glycoside-linked sugar chain and the 1-position of fucose are ⁇ -linked.
- Lentil lectin LC A Lientil Agglutinin from Lens Culinaris
- Endo bean lectin PSA Pea Lectin from Pisrnii sativum
- Broad bean lectin VF A Adgglutinin from Vicia faba
- Hyalurochawanta lectin AAL Aleuria aurant from Aleuria aurant
- the like are preferred.
- the antibody-producing cells of the present invention can be selected by culturing for a week, subculturing surviving cells or picking a colony, transferring it to another incubator, and continuing culturing in a lectin-containing medium. it can.
- the cells of the present invention target the gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose, and using the RDO method, for example, Can be made.
- cDNA or genomic DNA an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-doxy-GDP-mannose. Determine the base sequence of the prepared cDNA or genomic DNA.
- the target enzyme that is, the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto and 6-deoxy-GDP-mannose.
- yeast, animal cells, insect cells, plant cells, etc. have genes for enzymes that catalyze the dehydration reaction that converts the target GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose. Any of these can be used. Specific examples include the host cells described in 3. below.
- a method for preparing genomic DNA of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose is described, for example, in (a) of (1) above.
- Method for preparing genomic DNA described above.
- the DNA sequence of DNA is digested with an appropriate restriction enzyme and the like, cloned into a plasmid such as pBluescript SK (-) (Stratagene), and used in a commonly used nucleotide sequence analysis method, for example, Sanga et al.
- a reaction such as the dideoxy method [Procedings of the National Academy of Sciences (Proc. Natl. Acad. Sci., USA), 74, 5463 (1977)]
- the nucleotide sequence of the DNA can be determined by analysis using an automatic analyzer, for example, LF DNA Sequencer (Pharmacia).
- RD ⁇ can be prepared by a conventional method or by using a DNA synthesizer.
- RD ⁇ into host cells and select cells with mutations in the gene for the enzyme that catalyzes the dehydration reaction that converts the targeted enzyme, GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose
- Examples of the method include a method for directly detecting a mutation of a gene on a chromosome described in Molecular 'Cloning 2nd edition, Current Protocols in Molecular Biology, etc.
- the change in enzyme activity that catalyzes the dehydration reaction that converts GDP-mannose into GDP-4-keto and 6-deoxy-GDP-mannose as described in (a) of (1) above was used as an index. “Method for selecting a transformant” can also be used.
- the construction of RDO is described in Science, 273, 1386, (1996); Nichiya's Nature Medicine,, 285, (1998); Hepatology, 1462, (1997); —Gene Therapy, 5, 1960, (1999); Gene Therapy, 5, 1960,
- the cells of the present invention were prepared using the transposon system described in Nature Genet., 25, 35, (2000), and converting GDP-mannose to GDP-4-keto, 6-dexoxy-GDP-mannose.
- the cell of the present invention can be produced by selecting a mutant based on the activity of an enzyme that catalyzes a dehydration reaction that converts into a protein, or the sugar chain structure of a produced antibody molecule or a glycoprotein on a cell membrane as an index.
- the transposon system is a system that induces mutation by inserting a foreign gene into a chromosome at random, and usually uses the foreign gene contained in the transposon as a vector to induce mutation.
- a transposase expression vector for random insertion into the chromosome is simultaneously introduced into the cell.
- transposase Any transposase can be used as long as it is suitable for the sequence of the transposon to be used.
- any gene can be used as long as it induces mutation in the DNA of the cell.
- Cells such as yeast, animal cells, insect cells, and plant cells, have genes for enzymes that catalyze the dehydration reaction that converts the target GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose. Any of these can be used. Specific examples include the host cells described in 3 below.
- a method for selecting a mutant based on the activity of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose into GOT-4-keto, 6-doxy-GDP-mannose is described in, for example, One ) "Selecting transformants" using the change in enzyme activity that catalyzes the dehydration reaction to convert GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose as described in . ⁇
- the cells of the present study were constructed by introducing a mutation into the gene of an enzyme that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose, and Can be prepared by using a technique for selecting the cell strain of the present invention.
- enzymes that catalyze the dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose include GDP-mannose 4,6-dehydratase.
- any treatment can be used as long as it induces a point mutation, a deletion or a frame shift mutation in the DNA of the parent cell line.
- Specific examples include treatment with ethyl nitrosoparea, nitrosoguanidine, benzopyrene, and acridine dye, and irradiation with radiation.
- Various alkylating agents and carcinogens can also be used as mutagens. Examples of the method of causing a mutagen to act on cells include, for example, the third edition of tissue culture technology (Asakura Shoten), edited by The Japanese Society for Tissue Culture (1996), Nature Genet., 24, 314, (2000).
- Spontaneously occurring mutants include those that occur spontaneously by continuing subculture under normal cell culture conditions without special mutagenesis treatment. it can.
- a method for selecting a desired cell line based on a change in enzyme activity that catalyzes a dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose A method for selecting a desired cell line using the chain structure as an index and a method for selecting a desired cell line using the sugar chain structure of a glycoprotein on a cell membrane as an index include, for example, the method described in the above 1 (1).
- the “method for selecting a transformant” described in (a) using the change in enzyme activity that catalyzes the dehydration reaction of converting GDP-mannose to GDP-4-keto and 6-deoxy-GDP-mannose is used as an index. can give. ''
- transgenic non-human animal or plant or progeny thereof of the present invention The dehydration reaction of the present invention for converting GDP-mannose to GDP-4-keto, 6-dexoxy-GDP-mannose is described.
- Transgenic non-human animals or plants or their progeny in which the genomic gene has been modified to lack the activity of the catalyzing enzyme convert GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose It can be prepared from the embryonic stem cells, fertilized egg cells, or plant callus cells of the present invention prepared using the method described in 1. . -
- enzymes that catalyzes the dehydration reaction that converts GDP-mannose into GDP-4-keto, 6-deoxy-GDP-mannose include GDP-mannose 4,6-dehydratase.
- the target non-human animal for example, embryonic stem cells such as a horse, a sheep, a goat, a pig, a horse, a mouse, a rat, a chicken, a monkey, a rabbit, etc.
- embryonic stem cells of the present invention lacking the activity of the enzyme that catalyzes the deicing reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose. Can be.
- a gene encoding an enzyme that catalyzes a dehydration reaction that converts GDP-mannose on the chromosome into GDP-4-keto, 6-deoxy-GDP-mannose can be obtained by a known homologous recombination method [eg, , Nature, 326, 6110, 295 (1987), Cell, 51, 3, 503 (1987), etc.] and mutant clones inactivated or substituted with an arbitrary sequence.
- embryonic stem cells thus prepared for example, the mutant clone
- embryonic stem cell clones can be injected into the blastocyst (bi as tcys t) of a fertilized egg by chimera method or chimeric method.
- a chimeric individual comprising normal cells.
- a transgenic cell lacking the activity of an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose in whole body cells is obtained.
- Digenic non-human animals can be obtained.
- Target vectors for homologous recombination of target genes are described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Production of Mutant Mouse Using ES Cells (1995), etc. It can be prepared according to the method described.
- the target vector can be any of a replacement type, an insertion type, and a gene trap type.
- any method for introducing DNA into animal cells can be used.
- the electoporation method [site technology — (Cy to technol ogy), 3 , 133 (1990)], calcium phosphate method [Japanese Patent Application Laid-Open No. 2-227075], lipofection method [Procedindas ob 'The National' Academy 'Ob' Science (Proc. Natl. Ac ad. Sc i USA), 8>? 13 (1987)], injection method (manipulating 'mouse' Embrio 2nd edition), method using particle gun (gene gun) (Japanese Patent No. 2606856, Japanese Patent No.
- Methods for efficiently selecting homologous recombinants include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Production of mutant mice using ES cells (1995), etc. Positive selection, promoter selection, negative selection, poly A selection and the like can be used. Specifically, in the case of a target vector containing the hprt gene, after introduction into the embryonic stem cells lacking the hprt gene, the embryonic stem cells are cultured in a medium containing aminopterin, hypoxanthine and thymidine, and By selecting resistant strains, positive selection for selecting homologous recombinants containing the hprt gene can be performed.
- a target vector containing a neomycin resistance gene embryonic stem cells into which the vector has been introduced are cultured in a medium containing G418, and G418-resistant strains are selected to obtain homologous recombinants containing the neomycin resistance gene. You can choose.
- the target vector containing the DT gene a recombinant that is randomly inserted into the chromosome other than homologous recombination cannot grow due to DT toxicity because the DT gene is integrated into the chromosome and expressed.
- a fertilized egg When incorporating embryonic stem cells into a fertilized egg using the assembly chimera method, it is generally preferable to use a fertilized egg in a developmental stage before the 8-cell stage. When incorporating embryonic stem cells into a fertilized egg using the injection chimera method, it is generally preferable to use a fertilized egg in the stage of blastocyst development from the 8-cell stage. '' When a fertilized egg is transplanted into a female mouse, the fertilized egg obtained in a pseudopregnant female mouse in which fertilization has been induced is artificially transplanted and mated by mating with a vasectomized male non-human mammal.
- Pseudopregnant female mice can be obtained by natural mating, but fertilization is achieved by administering luteinizing hormone-releasing hormone (hereinafter abbreviated as LHRH) or an analog thereof and mating with male mice. Diet-induced pseudopregnant female mice can also be obtained.
- LHRH luteinizing hormone-releasing hormone
- fertilized egg cells of the target non-human animal for example, a porcupine, a sheep, a goat, a stag, a poma, a mouse, a rat, a chick, a monkey, a pupa, etc.
- the fertilized egg cell of the present invention can be produced which lacks the activity of an enzyme that catalyzes a dehydration reaction that converts -mannose into GDP-4-keto, 6-deoxy-GD-mannose.
- the prepared fertilized egg cells can be transferred to the embryos described in Manual Purification, Mouse, and Embryo 2nd Edition.
- the activity of an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto, 6-deoxy-GDP-mannose by transplanting into the oviduct or uterus of pseudopregnant females using the transplantation method and giving birth A transgenic non-human animal lacking the same can also be produced.
- the callus of the present invention in which the activity of the catalyzing enzyme is deleted can be produced.
- the prepared callus was auxinized according to a known method [tissue culture, 20 (1994); tissue culture, 21 (1995); Trend-in 'Trends in Biotechnology, 15, 45 (1997)]. And transgenes that lack the activity of an enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GD4-keto, 6-deoxy-GDP-mannose by culturing in a medium containing cytokinin and cytokinin. Plant can be produced.
- a method for producing a glycoprotein composition using the cells of the present invention will be described using the production of an antibody composition as a specific example.
- Antibody compositions are described in Molecular 'Cloning 2nd Edition, Current' Protocols 'in' Molecular, Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, 1988 (hereinafter Antibody's). Abbreviation), Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press, 1993 (hereinafter abbreviated as “monoclonal antipodes”), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press, 1996 (hereinafter, “antibody engineering”).
- the method can be obtained by, for example, expressing in a host cell into which a gene encoding an antibody molecule is introduced as follows, as described below.
- 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 cDNA into a suitable expression vector downstream of the promoter.
- a transformant producing the antibody composition of the present invention can be obtained.
- cDNA can be obtained from human or non-human animal tissues or cells using probe primers specific to the target antibody molecule in accordance with the "cDNA preparation method" described in (a) of (1) above. Can be prepared.
- yeast When yeast is used as a host cell, examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419) and the like.
- promoters of glycolytic genes such as hexoses, "PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter, MFal Promoters and CUP1 promoters.
- Examples of the host cell include microorganisms belonging to the genera Saccharomyces, Schizosaccharomyces, Krybetia spp., Trichosporon, Schuniomyces, etc., for example, Sacc aromyces cerevisiae, Sc izosaccharomyces pomb Kluyveromyces lactis, Trichosporon ullumyces all Can be given.
- any method can be used as long as it introduces DNA into yeast.
- the elect-mouth method [Methods. Enzymol., 194, 182 (1990)] ]
- the Spheroplast Method [Procedinations. Ob. The National 'Academy' Ob 'Science (Proc. Natl. Acad. Sci. USA), 84. 1929 (1978)]
- Lithium Toxic Acid Method [Journal of Bacteriology (J. Bacteriology), 153, 163 (1983)], Proceedings of the 'National Academy', 'Ob' Science (Proc. Natl. Acad. Sci. USA) ), 75, 1929 (1978)].
- examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), PAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (1990)]. ], PAS3-3 [Japanese Patent Laid-Open No. 2-227075], pCDM8 [Nature, 329, 840, (1987)], pcDNAI / Amp (Invitrogen), REP4 (Invitrogen), pAGE103 [Journal 'Pio chemistry (J. Biochemistry), 101, 1307 (1987)] and pAGE210. Any promoter can be used as long as it can be expressed in animal cells.
- the enhancer of the IE gene of human CMV may be used together with the promoter.
- Host cells include Namalwa cells, human cells, COS cells, monkey cells, CH0 cells, Chinese hamster cells, HBT5637 (JP-A-63-299), rat myeloma cells, and mouse myeloma cells. Examples include mackerel cells, Syrian hamster kidney-derived cells, embryonic stem cells, and fertilized egg cells.
- any method can be used as long as it is a method for introducing DNA into animal cells.
- Examples thereof include an electoral poration method [Cytotechnology, 3, 133 (1990)], Calcium phosphate method [Japanese Unexamined Patent Publication (Kokai) No. 2-227075], Lipofection method [Procedures 'ob' the 'national' academy 'op' science (Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)], Injection method [Manipulating 'Mouse' Emprio 2nd edition], Method using particle gun (gene gun) [Patent No. 2606856, Patent No.
- DEAE-dextran method Biomanual Series Gene introduction and expression ⁇ Analysis method (Yodosha) Takashi Yokota ⁇ Ken-ichi Arai (1994)] virus vector method [manipulating ⁇ ⁇ ⁇ mouse ⁇ embrio 2nd edition], etc. .
- the recombinant gene transfer vector and the PacuMouth virus are co-transfected into insect cells to obtain the recombinant virus in the culture supernatant of the insect cells. Can be expressed.
- Examples of the gene transfer vector used in the method include pVL1392, PVL1393, pBlueBacIII (all from Invitorogen) and the like.
- baculovirus for example, Autographacalifornica nuclear polyhedrosis virus, which is a virus that infects night moth family insects, can be used.
- Sf9 and Sf21 which are ovarian cells of Spodopterafrugiperda [current 'protocols-', 'more than a thousand Baculovirus Expression Vectors, A Laboratory Manual, I H. Freeman and Company, New York (1992)], and High5 (Invitrogen), which is an ovarian cell of Trichoplusiani, and the like can be used.
- Co-transfection methods of the above-described recombinant gene transfer vector and the above-described baculovirus into insect cells for preparing a recombinant virus include, for example, a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a ribofection method [Processing 'Ob the' National Academy of Science (Proc. Natl. Acad. Sci. USA), 84. 7413 (1987)].
- examples of the expression vector include a T1 plasmid and a tobacco mosaic virus vector.
- 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 geneactin 1 promoter.
- CaMV cauliflower mosaic virus
- Examples of the host itoda vesicle include plant cells such as tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, corn, and honeysuckle.
- Any method for introducing a recombinant vector can be used as long as it is a method for introducing DNA into plant cells.
- agrobacterium Agrobacterium [JP-A-59-140885, JP-A-60-70080, W094 / 00977], electroporation method [JP-A-60-251887], party A method using a glucan (gene gun) [Japanese Patent No. 2606856, Japanese Patent No. 2517813] and the like can be mentioned.
- a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression between Fc region and other proteins, etc. should be performed according to the method described in Molecular 'Cloning 2nd edition. Can be done.
- the antibody composition can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the antibody molecule in the culture, and collecting from the culture.
- the method for culturing the transformant in a medium can be performed according to a usual method used for culturing host cells.
- any medium that contains a carbon source, a nitrogen source, inorganic salts, and the like that can be used by the organism and can efficiently culture the transformant can be used.
- Either a natural medium or a synthetic medium may be used.
- the carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, and ethanol. And alcohols such as propanol.
- Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium or inorganic salts of organic acids, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn Chip liquor, casein hydrolyzate, soybean meal * 3 and soybean meal hydrolyzate, various fermented bacterial cells and digests thereof can be used.
- the inorganic salts potassium phosphate monobasic, potassium phosphate dibasic, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, man sulfate sulfate, sulfate class, calcium carbonate and the like can be used.
- the culture is usually performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
- the culture temperature is preferably 15 to 40 ° C, and the culture time is generally 16 hours to 7 days.
- the pH during culture is maintained at 3.0-9.0.
- the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
- an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture. '.
- a microorganism transformed with a recombinant vector using an inducible promoter as a promoter When culturing, an inducer may be added to the medium as needed. For example, when culturing a microorganism transformed in a recombinant vector using 1 ⁇ promoter overnight, a group using isop mouth pill- ⁇ -1 D-thiogalactovyranoside, etc. When culturing a microorganism transformed with the replacement vector, indoleacrylic acid or the like may be added to the medium.
- a medium for culturing a transformant obtained by using an animal cell as a host As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used RPMI 1640 medium [The Journal of the American Medical Association] [The Journal of the American Medical Association] ), 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 396 (1959)], 19 9 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], Whit ten medium [Development engineering experiments Manual-Transgenic Mouse Method (Kodansha) Katsuki Motoya (ed., 1987)] or a medium obtained by adding fetal calf serum or the like to these media.
- Culture is usually p H 6-8, 3 0-4 0 carried out under conditions such as 5% C0 2 presence 1-7 days. Culture can be carried out for one day to several months using
- antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
- TNM-FH medium As a medium for culturing a transformant obtained by using an insect cell as a host, generally used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies), ExCel l400, ExCel l405 (Both from JRH Biosciences), Grace's Insert Medium [Nature, 195, 788 (1962)] and the like can be used.
- Culture is usually performed at pH 6-7, 25-30. Perform for 1-5 days under conditions such as C.
- an antibiotic such as gentamicin may be added to the medium during the culture.
- a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ.
- a medium for culturing the transformant include commonly used Murashige and Skoog (MS) medium, white medium, or a medium to which plant hormones such as auxin and cytokinin are added. Can be used.
- Cultivation is usually performed at pH 5 to 9, 20 to 40 ° C for 3 to 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 recombinant vector into which DNA encoding an antibody molecule is incorporated is cultured according to a conventional culture method, and the antibody composition is determined.
- the antibody composition is produced by producing and accumulating a product, and collecting the antibody composition from the culture. Can.
- Methods for producing the antibody composition include a method of producing the antibody composition in a host cell, a method of secreting the antibody out of the host cell, and a method of producing the antibody composition on the outer membrane of the host cell.
- the method can be selected by changing the structure of the antibody molecule to be made.
- DNA encoding an antibody molecule and DNA encoding a signal peptide suitable for expression of an antibody molecule are introduced into an expression vector using a gene recombination technique, and the expression vector is used as the expression vector.
- the target antibody molecule can be actively secreted outside the host cell.
- the production amount can be increased by using a gene amplification system using a dihydrofolate reductase gene or the like.
- transgenic animal or plant by regenerating the cells of the transgenic animal or plant, an individual animal (transgenic non-human animal) or plant (transgenic plant) into which the gene has been introduced is created. It can also be used to produce an antibody composition.
- the transformant is an animal or plant individual
- the animal is bred or cultivated according to a usual method to produce and accumulate the antibody composition, and the antibody composition is collected from the animal or plant individual.
- the antibody composition can be produced.
- an antibody composition is produced and accumulated in the animal, and the antibody composition is collected from the animal.
- an antibody composition can be produced.
- Examples of the place of production and accumulation in the animal include milk (JP-A-63-309192) and eggs of the animal.
- any promoter that can be expressed in animals can be used.
- mammary cell-specific promoters such as ⁇ -casein promoter, / 3 casein promoter, j3 lactoglobulin promoter, whey acidic protein promoter and the like are preferably used.
- a transgenic plant into which DNA encoding an antibody molecule has been introduced can be prepared by a known method [tissue culture, ⁇ (1994); tissue culture, 21 (1995); Cultivated according to the trend “In” biotechnology (Trends in Biotechnolgy), 15, 45 (1997)] to produce and accumulate the antibody composition in the plant, and to produce the antibody composition from the plant.
- a method of producing an antibody composition by collecting the antibody can be mentioned.
- An antibody composition produced by a transformant into which a gene encoding an antibody molecule has been introduced is, for example, when the antibody composition is expressed in a lysed state in cells, after the culture is completed, the cells are centrifuged. After harvesting and suspending in an aqueous buffer, the cells are disrupted with an ultrasonic disrupter, French press, Mantongaulin homogenizer, Dynomill, etc. to obtain a cell-free extract.
- a normal enzyme isolation / 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, Getylaminoethyl (DEAE) -Sepharose, DIA.I0N HPA-75 (manufactured by Mitsubishi Chemical Co., Ltd.), anion exchange chromatography using a resin, S-Sepharose FF (Pharmacia), etc.
- a purified preparation of the antibody composition can be obtained by using a method such as electrophoresis such as isoelectric focusing or isoelectric focusing alone or in combination.
- the cells When the antibody composition is expressed by forming an insoluble substance in the cells, the cells are similarly collected, crushed, and centrifuged to collect the insoluble substance of the antibody composition as a precipitate fraction.
- the recovered insoluble form of the antibody composition is solubilized with a protein denaturant. After diluting or dialyzing the solubilized solution to return the antibody composition to a normal three-dimensional structure, a purified sample of the antibody composition can be obtained by the same isolation and purification method as described above.
- the antibody composition When the antibody composition is secreted extracellularly, the antibody composition can be collected in the culture supernatant. That is, a soluble fraction is obtained by treating the culture by a method such as centrifugation as described above, and the antibody composition is purified from the soluble fraction by using the same isolation and purification method as described above. You can get a sample.
- Examples of the antibody composition thus obtained include an antibody, an antibody fragment, and a fusion protein having an Fc region of the antibody.
- the humanized antibody expression vector is an expression vector for animal cells into which genes encoding the heavy chain (H chain) and light chain (L chain) C regions of human antibodies have been incorporated. It can be constructed by cloning the genes encoding the H chain, L chain, and C region of the human antibody into an expression vector.
- the C region of the human antibody may be any of the H chain and L chain C regions of any human antibody.
- the C region of the IgGl subclass of the H chain of the human antibody hereinafter referred to as hCrl
- the human antibody And the C region of the ⁇ class of the L chain (hereinafter referred to as hC /).
- chromosomal DNA consisting of exon and intron can be used, and cDNA can also be used.
- Any expression vector for animal cells can be used, so long as it can incorporate and express a gene encoding the C region of a human antibody.
- PAGE107 [Cy to technology, 3, 133 (1990)]
- pAGE103 [Journal of Biochemistry., 101, 1307 (1987)]
- pHSG274 [Gene (Gene), 27, 223 (1984)]
- pKCR Proceedings-Ob the National 'Academy' Ob 'Science (Pro Natl. Acad. Sci. USA), 78, 1527 (1981)]
- pSGl ⁇ d2-4 [Cytotechnology, 4, 173 (1990)] and the like.
- the promoter and enhancer used in the expression vector for animal cells include the initial promoter of SV40 and the enhancer [Journal of Biochemistry (J.
- the vector for expressing a humanized antibody can be either a type in which the antibody H chain and the L chain are present on separate vectors, or a type in which the antibody is present on the same vector (hereinafter referred to as tandem type). Ease of construction of humanized antibody expression vector that can be used, ease of introduction into animal cells, balance of antibody H chain and L chain expression levels in animal cells, etc. To J. Immunol. Methods (J. Immunol. Methods, j_67, 271 (1994)).
- the constructed humanized antibody expression vector can be used for expression of a human chimeric antibody and a human CDR-grafted antibody in animal cells.
- cDNA encoding V region of antibody from non-human animal
- a cDNA encoding an antibody of an animal other than a human, for example, the H chain and L chain V regions of a mouse antibody can be obtained as described below.
- MRNA is extracted from the eight hybridoma cells producing the desired mouse antibody, and the synthesized cDNA for synthesizing the cDNA is cloned into a vector such as a phage or plasmid to prepare a cDNA library.
- a vector such as a phage or plasmid to prepare a cDNA library.
- a recombinant phage or a recombinant plasmid having a cDNA encoding the H chain V region and a cDNA encoding the L chain V region were obtained.
- the isolated recombinant phage or recombinant plasmid is isolated.
- the entire nucleotide sequence of the H chain and L chain V region of the target mouse antibody on the recombinant phage or recombinant plasmid is determined, and the entire amino acid sequence of the H chain and L chain V region is deduced from the nucleotide sequence.
- any animal can be used, such as mice, rats, hamsters, and egrets, as long as they can produce hybridoma cells.
- Methods for preparing total RNA from hybridoma cells include the guanidine thiocyanate-trifluoride cesium method [Methods in Enzymol., 154, 3 (1987)]
- the method for preparing mRNA includes an oligo (dT) -immobilized cellulose column method [Molecular Cloning: A Laboratory Manual], Cold Spring Harbor Lab. Press New York, 1989 ] And the like.
- kits for preparing mRNA from hybridoma cells include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.
- any vector can be used as a vector into which a cDNA synthesized by using DiRJ ⁇ extracted from the hybridoma cells as type II can be inserted, as long as it can incorporate the cDNA.
- ZAP Express [Strategies' (Strategies), 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research],, 9494 (1989)], Azap II (Stratagene) , AgtlO, Agtll [DNA Cloning: A Practical Approach], I, 49 (1985)], Lambda BlueMid (CI on tech), AExCelU pT7T3 18U (Pharmacia Co., Ltd.), pcD2 [Molecular and Cellular Biology (Mol.
- Escherichia coli for introducing a cDNA library constructed from a phage or plasmid vector, any Escherichia coli capable of introducing, expressing and maintaining the cDNA library can be used.
- Escherichia coli capable of introducing, expressing and maintaining the cDNA library can be used.
- Methods for selecting cDNA clones encoding the H chain and L chain V regions of non-human animal antibodies from the cDNA library include colony hybridization using an isotope or a fluorescently labeled probe, or The method can be selected by the plaque hybridization method [Molecular Cloning: a. Laholatri ..] (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Lab. Press New York, 1989.
- primers are prepared, and a cDNA or a cDNA library synthesized from mRNA is used as a type III for polymerase chain reaction [hereinafter, referred to as PCR method; Molecular cloning: A ⁇ Laboratory 1 ⁇ Manual (Molecular Cloning: A Laboratory Manual) ), Cold Spring Harbor Lab. Press New York, 1989; cDNA encoding H-chain and L-chain V regions was obtained according to Current Protocols in Molecular Biology, Supplement 1-34]. It can also be prepared. .
- the cDNA selected by the above method is cleaved with an appropriate restriction enzyme and the like, cloned into a plasmid such as Bluescript SK (-) (manufactured by Stratagene), and subjected to a commonly used nucleotide sequence analysis method, for example, Sanger. And the dideoxy method [Procedings of the National Co., Ltd., of Science (Proc. Natl. Acad. Sci., USA), 74, 5463 (1977)].
- the nucleotide sequence of the cDNA can be determined by analysis using an automatic nucleotide sequence analyzer, for example, ALF DNA Sequencer (Pharmacia).
- the entire amino acid sequence of the H chain and L chain V regions was estimated, and the entire amino acid sequence of the H chain and L chain V regions of the known antibody [Sequences of protein, proteins, immunological and By comparing the sequence of the obtained cDNA with the complete amino acid sequence of the H-chain and L-chain V regions of the antibody, including the secretory signal sequence, by comparing with the sequence (Sequences of Proteins of Immunologicalliiterest), US Dept. Health and Human Services, 1991]. You can check if you are coding.
- the entire amino acid sequence of the H and L chain V regions of the body including the secretory signal sequence the entire amino acid sequence of the H and L chain V regions of a known antibody [Sequences. Im Roe Deer ⁇ / ⁇ Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 1991]
- the length of the secretory signal sequence and the N-terminal amino acid sequence can be estimated, and the subgroup to which they belong can be known.
- the amino acid sequences of the CDRs of the H chain and the L chain V region are also known from the amino acid sequences of the H chain and the L chain V region of the known antibody [Sequences of proteins, proteins, immunological interest ( Sequences of Proteins of Immunological Interest), US Dept. Health and Human Services, 1991].
- the H-chain and L-chain V regions of a non-human animal antibody are located upstream of the gene encoding the H-chain and L-chain C regions of the human antibody in the humanized antibody expression vector described in 3 (1) of this section.
- a human-type chimeric antibody expression vector can be constructed.
- the cDNA encoding the H chain and L chain V regions of the antibody of a non-human animal can be obtained by combining the nucleotide sequence of the 3 ′ end of the antibody and L chain V regions of the non-human animal with the ⁇ chain of the human antibody.
- the humanized antibody according to item (1) of item 3 of the present invention which is composed of a base sequence at the 5 'end of the C region of the L chain and ligated to a synthetic DNA having a recognition sequence for an appropriate restriction enzyme at each end.
- a human-type chimeric antibody expression vector can be constructed by cloning upstream of the gene encoding the human antibody L chain and L chain C region of the expression vector so that they can be expressed in an appropriate form.
- CDNA encoding the H chain and L chain V regions of the human CDR-grafted antibody can be constructed as follows. First, the amino acid sequence of the framework (hereinafter referred to as FR) of the H chain and L chain V regions of the human antibody to be transplanted with the CDRs of the H chain and L chain V regions of the target non-human animal antibody is selected. . As the amino acid sequence of FR in the H chain and L chain V regions of a human antibody, any amino acid sequence can be used as long as it is derived from a human antibody.
- amino acid sequence of the FRs of the H chain and L chain V regions of the human antibody registered in databases such as Protein Data Bank, and the common subgroups of the FRs of the H region and L chain V region of the human antibody FR Amino acid sequence [Sequences of Proteins of Immunology-Immunological Interest, US Det. Health and Human Services, 1911], etc.
- ⁇ the amino acid sequence of the FRs of the H-chain and L-chain V regions of the antibody of the target non-human animal as much as possible It is desirable to select an amino acid sequence having high homology (at least 60% or more).
- the amino acid sequence of the CDRs of the H chain and L chain V region of the target non-human animal antibody is transplanted into the FR amino acid sequence of the H chain and L chain V region of the selected human antibody.
- the designed amino acid sequence uses the codons found in the nucleotide sequence of the antibody gene [Sequences of Proteins * Immunological] [Sequences of Proteins of Immunological Interest], US Dept. Heal th and Human Services, 1991]
- the DNA sequence is converted into a DNA sequence in consideration of the above, and a DNA sequence encoding the amino acid sequence of the H chain and L chain V regions of the human CDR-grafted antibody is designed.
- the amplified product is cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), the nucleotide sequence is determined by the method described in (2) of Section 3 of this section, and the desired human CDR-grafted antibody is obtained.
- a plasmid having a DNA sequence encoding the amino acid sequence of the H chain and L chain V regions is obtained.
- the human-type CDR-grafted antibody in which the CDR of the H chain and L chain V region of the antibody of the target non-human animal is simply linked to the FR of the H chain and L chain V region of the human antibody has its antigen-binding activity Is known to be lower than that of the original non-human animal antibody [Bio / Technology (BI0 / TECHN0L0GY), 9, 266 (1991)]. This is due to the fact that not only CDRs but also some amino acid residues of FRs directly or indirectly participate in antigen-binding activity in the V region of the original non-human animal antibody.
- human-type CDR-grafted antibodies include amino acid residues that are directly involved in antigen binding in the amino acid sequence of FRs in the H chain and L chain V regions of the human antibody.
- Amino acid residues that interact with the amino acid residues maintain the three-dimensional structure of the antibody, identify amino acids residues that are indirectly involved in antigen binding, etc. It has been attempted to increase the decreased antigen-binding activity by modifying the amino acid residues found in the antibody of the animal of Japan [Bio / Technology-1 (BI0 / TECHN0L0GY), 266 (1991)].
- the modification of FR amino acid residues in the H chain and L chain V regions of a human antibody can be achieved by performing the PCR method described in item 3 (5) of this section using the synthetic DNA for modification. Determine the nucleotide sequence of the amplified product after PCR by the method described in (2) of this section 3 and confirm that the target modification has been performed. (7) Construction of human CDR-grafted antibody expression vector
- the construct was constructed by (5) and (6) in Section 3 upstream of the gene encoding the H chain and L chain C regions of the human antibody.
- a cDNA encoding the H-chain and L-chain V regions of the human GDR-grafted antibody can be cloned to construct a human CDR-grafted antibody expression vector.
- the synthetic DNA used to construct the H chain and L chain V regions of the human CDR-grafted antibody in (5) and (6) of section 3 of this section the 5 ′ end of the synthetic DNA located at both ends is used.
- a recognition sequence for an appropriate restriction enzyme By introducing a recognition sequence for an appropriate restriction enzyme, they can be placed upstream of the gene encoding the H chain and L chain C region of the human antibody in the humanized antibody expression vector described in item 3 (1) of this section. By cloning such that it can be expressed in various forms, a human CDR-grafted antibody expression vector can be constructed.
- humanized antibodies By introducing the humanized antibody expression vector described in (4) and (7) of this section 3 into appropriate animal cells, a human chimeric antibody and a human CDR-grafted antibody (hereinafter collectively referred to as humanized antibodies) ) Can be obtained in a stable manner.
- Examples of a method for introducing a humanized antibody expression vector into animal cells include an electoral poration method [Japanese Patent Laid-Open No. 2-257891; Cytoteclinology, 3, 133 (1990)] and the like.
- any animal cell that can produce a humanized antibody can be used.
- mice is derived from mouse myeloma NS0 cells, SP2 / 0 cells, Chinese hamster ovary cells CHO / dhfr-cells, CH0 / DG44 cells, rat myeloma YB2 / 0 cells, IR983F cells, and Syrian eight Muster kidney.
- Certain BHK cells, Namalba cells, which are human myeloma cells, and the like are preferable, and preferred are the Chinese hamster ovary cells, CH0 / DG44 cells, rat myeloma ⁇ 2 / 0 cells, and the cells described in 1.
- a transformant that stably produces a humanized antibody can be produced using G418 sulfate (hereinafter referred to as G418; manufactured by SIGMA) according to the method disclosed in JP-A-2-2577891. ) Can be selected depending on the animal cell culture medium containing the drug.
- G418 G418 sulfate
- Animal cell culture media include RPMI1640 medium (Nissui Pharmaceutical), GIT medium (Nippon Pharmaceutical), EX-CELL302 medium (JRH), IMDM medium (GIBC0 BRL), Hybridoma-SFM medium (Manufactured by GIBCO BRL), or a medium in which various additives such as fetal bovine serum (hereinafter referred to as FBS) are added to these media.
- FBS fetal bovine serum
- the amount of humanized antibody produced in the culture supernatant and the antigen-binding activity are determined by enzyme-linked immunosorbent assay [hereinafter referred to as ELISA; Antibodies: A Laboratory 'Manual], Cold Spring Harbor Laboratory, Chapter 14 , 1998, Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996].
- ELISA enzyme-linked immunosorbent assay
- the transformant can increase the amount of humanized antibody produced using a DHFR gene amplification system or the like according to the method disclosed in Japanese Patent Application Laid-Open No. 2-2587891.
- Humanized antibodies can be purified from the culture supernatant of the transformant using a protein A column [Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 8, 1988, Monoclonal Antibodies: Principles and Practice, Academic Press Limited, 1996].
- other purification methods usually used for protein purification can be used.
- purification can be performed by a combination of gel filtration, ion exchange chromatography, and ultrafiltration.
- the molecular weight of the H-chain, L-chain of the purified humanized antibody or the entire antibody molecule can be determined by polyacrylamide gel electrophoresis [hereinafter referred to as SDS-PAGE; Nature, 227, 680 (1970)] or Western blotting.
- SDS-PAGE polyacrylamide gel electrophoresis
- An expression vector for an Fc fusion protein is an expression vector for animal cells into which a gene encoding a protein to be fused with the Fc region of a human antibody is incorporated. It can be constructed by cloning a gene coding for the protein to be fused with the protein.
- the Fc region of a human antibody includes a region containing the CH2 and CH3 regions, as well as a region containing the hinge region and a part of CH1. Any substance may be used as long as at least one amino acid of CH2 or CH3 has been deleted, substituted, added or inserted, and has substantially the binding activity to the Fcr receptor.
- the gene encoding the protein to be fused with the Fc region of the human antibody chromosomal DNA consisting of exon and intron can be used, and cDNA can also be used.
- the PCR method ( recruiter-I 'cloning 2nd edition; Current Protocols in Molecular Biology, Supplement 1-34) was used for each gene sequence as type II. Things to do.
- any vector can be used as long as it can integrate and express a gene encoding the C region of a human antibody.
- pAGE107 [Cy to technology, 3, 133 (1990)]
- pAGE103 [Journal of Biochemistry (J. Biochem.), 101, 1307 (1987)]
- pHSG274 [Gene ), 27, 223 (1984)]
- pKCR Procedure Ding's of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 78, 1527 (1981)]
- pSGl / 3d2-4 [Cytotechnology, 4, 173] (1990)].
- Promoters and enhancers used in animal cell expression vectors include the SV40 early promoter and enhancer [Journal 'ob' Biochemistry., 101, 1307 (1987)], Moroni LTR of mouse leukemia virus [Biochemical. Biophys. Res. Commun., 149, 960 (1987)], promoter of immunoglobulin heavy chain [Cell ), 41, 479 (1985)] and enhancers [Cell, 33-717 (1983)].
- DNA encoding the protein to be fused with the Fc region of a human antibody can be obtained as follows. '
- MRNA is extracted from cells or tissues expressing the protein to be fused with the desired Fc, and cDNA is synthesized.
- the synthesized cDNA is cloned into a vector such as a phage or a plasmid to prepare a cDNA library.
- a recombinant phage or a recombinant plasmid having a cDNA encoding the target protein is isolated using the gene sequence portion of the target protein as a probe.
- the entire nucleotide sequence of the target protein on the recombinant plasmid is determined, and the entire amino acid sequence is estimated from the nucleotide sequence. '
- mice As animals other than humans, any animal such as mice, rats, hamsters, and egrets can be used as long as cells and tissues can be extracted therefrom.
- Methods for preparing total RNA from cells and tissues include the guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzyniol., 154, 3 (1987)], and the preparation of mRNA from total RNA.
- Examples of the method include an oligo (dT) -immobilized cellulose column method (Molecular Cloning, Second Edition) and the like.
- kits for preparing mRNA from cells and tissues include Fast Track mRNA Isolation Kit (Invitrogen), Quick Prep mRNA Purification Kit (Pharmacia) and the like.
- Methods for synthesizing cDNA and preparing a cDNA library include conventional methods (Molecular Cloning, Second Edition; Current Protocols, Molecular Molecular Biotechnology, Supplement 1-34), or commercially available methods.
- the kit include a method using a Super Script TM Plasmid System for cDNA Synthesis and Plasmid Cloning (GIBCO BRL) and a ZAP-cDNA Synthesis Kit (Stratagene).
- any vector can be used as a vector into which a cDNA synthesized as a type II mRNA extracted from cells or tissues can be inserted, as long as the vector can incorporate the cDNA.
- ZAP Express [Strategies, 5, 58 (1992)]
- pBluescript II SK (+) [Nucleic Acids Research, 17, 9494 (1989)]
- AzapII (Stratagene)
- AgtlO Agtll
- Agtll DNA Cloning: A 'Practical.
- any Escherichia coli can be used as long as the cDNA library can be introduced, expressed and maintained.
- XL 1 -Blue RF ' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088, Y1090 [Science, 222, 778] (1983)], NM522 [Journal of Molecular Biology (J. Mol. Biol.), 166, 1 (1983)], K802 [Journal of Molecular Biology (J. Mol. Biol.), 16, 118 (1966)] and JM105 [Gene, 38, 275 (1985)].
- the colony hybridization method or the hybrid hybridization method (molecular method) using an isotopic or fluorescently labeled probe is used. 'Cloning 2nd edition).
- a primer can be prepared, and a cDNA or cDNA library synthesized from mRNA can be used as a type II to prepare a cDNA encoding a target protein by a PCR method.
- a PCR method can be used as a method for fusing the target protein with the Fc region of a human antibody.
- an arbitrary synthetic oligo DNA (primer) is designed on the 5 ′ side and 3 ′ side of the gene sequence of the target protein, and a PCR method is performed using the primer to obtain a PCR product.
- an arbitrary primer is designed for the gene sequence of the Fc region of the human antibody to be fused, and a PCR product is obtained.
- primers are designed so that the same restriction enzyme site or the same gene sequence exists on the 3 'side of the PCR product of the protein to be fused and the 5' side of the PCR product of the Fc region.
- the mutation is introduced by using a primer into which the mutation has been introduced. Further PCR is performed using the obtained two types of PCR fragments to ligate both genes. Alternatively, the ligation can be carried out by ligation after the same restriction enzyme treatment.
- the gene sequence linked by the above method is digested with an appropriate restriction enzyme or the like, and then cloned into a plasmid such as pBluescript SK (-) (manufactured by Stratagene), and a commonly used nucleotide sequence analysis method, for example, Sanger The dideoxy method [Procedings of the National Aca Demi-Io-ob 'Science (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABI PRISM377 DNA sequencing
- the base sequence of the DNA can be determined by analysis using a base sequence analyzer such as PE Biosystems. Estimate the entire amino acid sequence of the Fc fusion protein from the determined base sequence and compare it with the target amino acid sequence to confirm that the obtained cDNA encodes the complete amino acid sequence of the Fc fusion protein including the secretory signal sequence can do. '
- any animal cell that can produce the Fc fusion protein can be used.
- W NS0 cells, SP2 / 0 cells, mouse myeloma cells, Chinese Eight Mus. Ovary cells, CHO / dhfr-cells, CH0 / DG44 cells, rat myeloma YB2 / 0 cells, IR983F cells BHK cells derived from Syrian hamster kidney, Namalba cells which are human myeloma cells, and the like, preferably, CH0 / DG44 cells, Chinese hamster ovary cells, rat myeloma YB2 / 0 cells, and Host cells and the like used in the method of the present invention can be mentioned.
- a transformant that stably produces the Fc fusion protein can be selected using an animal cell culture medium containing a drug such as G418 according to the method disclosed in JP-A-2-2577891.
- an animal cell culture medium containing a drug such as G418 As a medium for cultivation of animal cysts, RMI 1640 medium (Nissui Pharmaceutical), GIT medium (Nippon Pharmaceutical), EX-CELL302 medium (JRH), IMDM medium (GIBCO BRL), Hybridoma -SFM medium (manufactured by GIBCO BRL), or a medium obtained by adding various additives such as fetal calf serum to these mediums can be used.
- Fc fusion protein By culturing the obtained transformant in a medium, Fc fusion protein can be produced and accumulated in the culture supernatant.
- the production amount and antigen-binding activity of the Fc fusion protein in the culture supernatant can be measured by the ELISA method or the like.
- the transformed strain can increase the production of the Fc fusion protein using a dhir gene amplification system or the like according to the method disclosed in Japanese Patent Application Laid-Open No. 2-25789U.
- the Fc fusion protein can be purified from the culture supernatant of the transformed strain using Protein A or Protein G (Antibodies, Chapter 8, Monoclonal Antipodes).
- a purification method usually used for protein purification can be used.
- purification can be performed by a combination of gel filtration, ion exchange chromatography, ultrafiltration, and the like.
- the molecular weight of the whole purified Fc fusion protein molecule can be determined by SDS-PAGE [Nature, 227, 680 (1970)] or Western blotting method (Antibodies, Chapter 12, Monochrome, Seventy bodies), etc. Can be measured.
- the method for producing a baby using an animal cell as a host has been described. However, as described above, it can be produced in a bacterium, yeast, insect cell, plant cell or animal or plant individual. In the case of cells already having the ability to express a glycoprotein such as an antibody molecule, the production cells are prepared using the method described in 1., and then the cells are cultured. By purifying the target antibody or glycoprotein, the antibody composition or glycoprotein of the present invention can be produced.
- the protein content of the purified glycoprotein composition includes Current Protocols In Protein Science, John Wiley & Sons Inc., (1995), The Japanese Biochemical Society.
- a purified glycoprotein composition is labeled with a compound such as radioisotope, and the binding activity of the labeled glycoprotein composition to a receptor or an interacting protein is measured.
- a compound such as radioisotope
- the interaction between protein and protein can also be measured using various instruments such as iacore's BIAcore series (J. I Maraud nunol. Methods, 145, 229 (1991), Experimental Medicine SiJ Biomanual UP Series) Experimental method for protein molecular interaction, Yodosha (1996)).
- a detection method in which a method of detecting a labeled substance and an antibody-antigen reaction using an antibody specific to a glycoprotein composition to be detected are preferably used.
- the binding activity between the antibody composition and the antigen, the binding activity of the antibody composition to an antigen-positive cultured cell line, etc. are determined by ELISA and fluorescent antibody method.
- the safety and therapeutic effect of the antibody composition in humans can be evaluated using an appropriate model of an animal species relatively close to humans, such as a cynomolgus monkey.
- the sugar chain structure of the glycoprotein composition expressed in various cells can be determined according to the usual analysis of the sugar chain structure.
- the sugar chains bound to the IgG molecules in the antibody composition are composed of neutral sugars such as galactose, mannose, and fucose, amino sugars such as N-acetyl darcosamine, and acidic sugars such as sialic acid. It can be performed using techniques such as sugar composition analysis and sugar chain structure analysis using the two-dimensional sugar chain map method.
- composition analysis of the sugar chain of the glycoprotein composition neutral sugar or amino sugar is released by acid hydrolysis of the sugar chain with trifluoroacetic acid or the like, and the composition ratio can be analyzed.
- BioLC sugar composition analyzer
- HPAEC-PAD high performance anion-exchange chromatography-pulsed amperometric detection
- composition ratio can also be analyzed by a fluorescent labeling method using 2-aminopyridine. Specifically, a known method [agricultural and biological chemistry]
- glycoproteins are degraded by hydrazine to release sugar chains, and fluorescent labeling of sugar chains with 2-aminopyridine (hereinafter abbreviated as PA) [Journal of Biochemistry (J. Biochem.) , 95, 197 (1984)], the sugar chain is separated from excess PA-forming reagent and the like by gel filtration, and reversed-phase chromatography is performed. Next, normal phase chromatography is performed on each peak of the separated sugar chains. Based on these results, the results were plotted on a two-dimensional glycan map and compared with the glycan standard (TaKaRa) and the literature [Analytical Biochem., 171, 73 (1988)]. Sports The sugar chain structure can be estimated from the comparison of the plots. ,
- mass spectrometry such as MALDI-T0F-MS of each sugar chain can be performed to confirm the structure estimated by the two-dimensional sugar chain map method.
- the glycoprotein composition of the present invention has a sugar chain structure to which fucose is not bound, for example, the affinity of the glycoprotein composition with a receptor is improved. Effects such as improvement in half-life, improvement in tissue distribution after administration of the glycoprotein composition into the blood, and improvement in interaction with proteins required for pharmacological activity can be expected.
- the antibody composition of the present invention has a high effector function, that is, an antibody-dependent cytotoxic activity.
- glycoprotein composition having high physiological activity is useful in the prevention and treatment of various diseases including cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, and viral or bacterial infections. It is.
- cancer cells are growing.
- Ordinary anticancer drugs are characterized by inhibiting the growth of cancer cells.
- an antibody having a high antibody-dependent cytotoxicity can treat cancer by damaging cancer cells by a cell-killing effect, and is therefore more effective as a therapeutic drug than ordinary anticancer drugs.
- the antitumor effect of antibody drugs alone is often insufficient at present, and combination therapy with chemotherapy is performed [Science, 1197, 1998]. A stronger anti-tumor effect of the produced antibody composition alone would reduce reliance on chemotherapy and lead to reduced side effects.
- immune diseases such as inflammatory diseases, autoimmune diseases, and allergies
- in vivo reactions in those diseases are triggered by the release of mediator molecules by immune cells.
- allergic reactions can be suppressed by removing immune cells using an antibody having high antibody-dependent cytotoxicity.
- Cardiovascular diseases include arteriosclerosis. Atherosclerosis is currently treated with a balloon catheter, but cardiovascular disease is prevented by suppressing the proliferation of arterial cells during restenosis after treatment with antibodies that have high antibody-dependent cytotoxicity. And can be treated.
- An antibody that recognizes a tumor-associated antigen an antibody that recognizes an antigen associated with allergy or inflammation, an antibody that recognizes an antigen that is associated with a cardiovascular disease, an antibody that recognizes an antigen that is associated with an autoimmune disease, or a virus or bacterium
- Specific examples of antibodies that recognize antigens associated with infection are described below.
- Antibodies that recognize tumor-associated antigens include anti-GD2 antibody (Anticancer Res., 13, 331, 1993), GD3 antibody (Cancer Immunol. Immunother., 36> 260, 1993), anti-GM2 antibody (Cancer Res., 54, 1511, 1994), anti-HER2 antibody (Proc. Natl. Acad. Sci.'USA, 89, 4285, 1992), anti-CD52 antibody (Nature, 332, 323, 1988), anti-MAGE antibody (British J.
- Anti-HMl.24 antibody Molecular I maraudal unol., 36, 387, 1999
- Anti-parathyroid hormone-related protein (PTH r P) antibody (Cancer, 88, 2909, 2000), anti-FGF8 antibody (Proc. Natl. Acad. Sci. USA, 86, 9911, 1989)
- anti-PMSA antibody J. Urology, 160, 2396, 1998)
- anti-vascular endothelium Cell growth factor antibody Cancer Res., 57, 4593, 1997) or anti-vascular endothelial cell growth factor receptor antibody (Oncoge ne, 19, 2138, 2000)
- anti-CA125 antibody anti-17-1A antibody, anti-integrin o
- vi33 antibody, anti-CD33 antibody, anti-CD22 antibody, anti-HLA antibody, anti-HLA-DR antibody, anti-CD20 antibody anti- Examples include the CD19 antibody, anti-EGF receptor antibody (I band unolgy Today, 21, 03, 2000), and anti-CD10 antibody (American Journal of Clinical Pathology, 113, 374, 2000).
- Antibodies that recognize antigens associated with allergy or inflammation include anti-interleukin-6 antibody (I band unol. Rev., 127, 5, 1992) and anti-interleukin-6 receptor antibody (Molecular Immunol., 31). 371, 1994), anti-interleukin 5 antibody (Immunol. Rev., 127, 5, 1992), anti-interleukin 5 receptor antibody, anti-interleukin 4 antibody (Cytokine, 3, 562, 1991), Interleukin 4 receptor anti- #: (J. Immunol.
- anti-tumor necrosis factor antibody Hybridoma, 13, 183, 1994
- anti-tumor necrosis factor receptor antibody Molecular Pharmacol
- anti-CCR4 antibody Nature, 400, 776, 1999
- anti-chemokine antibody J. Immunol. Meth., IU, 249, 1994
- anti-chemokine receptor # Antibody (J. Exp. Med., 186, 1373, 1997), anti-IgE antibody, anti-CD23 antibody, anti-CDlla antibody (Immunology Today, 21, 403, 2000), anti-CRTH2 antibody (J. Immunol., 162, 1278, 1999), Anti-CCR8 antibody (W099 / 25734), anti-CCR3 antibody (US6207155) and the like.
- Antibodies that recognize antigens associated with cardiovascular diseases include anti-GpIIb / IIIa antibodies (J. Immunol., 152, 2968, 1994), anti-platelet-derived growth factor antibodies (Science, 253, 1129, 1991), Platelet-derived proliferation factor receptor antibody (J. Biol. Cheni., 272, 17400, 1997) or anticoagulation factor antibody (Circulation, 101, 1158, 2000).
- Antibodies that recognize antigens associated with autoimmune diseases include anti-self thigh antibodies (Immunol-Letters, 72, 61, 2000), anti-CDlla antibody, anti-ICAM3 antibody, anti-CD80 antibody, anti-CD2 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-integrin ⁇ 4187 antibody, anti-CD4Q.L antibody, anti-IL -2 receptor antibody (Immunology Today, 21, 403, 2000).
- Antibodies that recognize antigens associated with viral or bacterial infection include anti-gp12 antibodies (Structure, 8, 385, 2000), anti-CD4 antibody (J. Rheumatology, 25, 2065, 1998), anti-CCR4 antibody, anti-verotoxin antibody (J. Clin. Microbiol., 37, 396, 1999) .
- the above antibodies can be obtained from public institutions such as ATCC (The American Type Culture Collection), RIKEN Cell Development Bank, Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology, or Dainippon Pharmaceutical Co., Ltd., R & D SYSTEMS It can be obtained from private reagent sales companies such as PharMingen, Cosmo Bio, and Funakoshi.
- the pharmaceutical containing the glycoprotein composition of the present invention can be administered alone as a therapeutic agent, it is usually mixed with one or more pharmacologically acceptable carriers, and It is desirable to provide as a pharmaceutical preparation produced by any method well-known in the technical field of pharmaceutics.
- intravenous administration can be preferably used.
- Dosage forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like. '
- Formulations suitable for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
- Liquid preparations such as emulsions and syrups include water, sugars such as sucrose, sorbitol, fructose, glycols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil, soybean oil, p- It can be produced using preservatives such as hydroxybenzoic acid esters, flavors such as strawberry flavor, and ⁇ ⁇ permint as additives.
- Capsules, tablets, powders, granules, etc. are excipients such as lactose, glucose, sucrose, mannitol, disintegrants such as starch, sodium alginate, lubricants such as magnesium stearate, talc, polypier alcohol, It can be produced using a binder such as hydroxypropylcellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin as additives.
- Formulations suitable for parenteral administration include injections, suppositories, sprays and the like.
- the injection is prepared using a carrier comprising a salt solution, a glucose solution, or a mixture of both.
- a powdered injection can be prepared by freeze-drying a humanized antibody according to a conventional method and adding sodium chloride thereto.
- Suppositories are prepared using carriers such as cocoa luster, hydrogenated fat or carboxylic acid.
- Sprays are prepared using the compound itself or a carrier that does not irritate the oral and respiratory tract mucosa of the recipient, and disperses the compound as fine particles to facilitate absorption.
- the carrier include lactose and glycerin.
- properties of the compound and carrier used thus, preparations such as air opening sleet and dry powder are possible.
- the components exemplified as additives for oral preparations can also be added.
- the dose or frequency of administration varies depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but is usually 10 ig / kg to 20 mg / kg per day for an adult.
- methods for examining the antitumor effect of the antibody composition on various tumor cells include CDC activity measurement methods and ADCC activity measurement methods in in vitro experiments, and mouse and other methods in in vivo experiments. Examples include antitumor experiments using a tumor system in test animals.
- FIG. 1 is a diagram showing ADCC activity of purified 2® anti-CCR4 human chimeric antibodies.
- the vertical axis shows the cytotoxic activity, and the horizontal axis shows the antibody concentration.
- Transformed strain obtained from knockout strain of GDP-mannose 4,6-dehydratase gene CHO SM strain SM3G1 / CCR4 antibody produced by SM3G1 / CCR4 strain, Transformation obtained from parental CH0 / DG44 cells The activity of the CH0 / CCR4 antibody produced by the strain CH0 / CCR4 is shown. .
- FIG. 2 shows the viable cell density (A) and cell viability (B) when serum-free Fued batch culture was performed using the SM3G1 / CCR4-AF and CH0 / CCR4-AF strains conditioned to serum-free medium.
- FIG. 4 is a graph showing changes in antibody concentration (C). The horizontal axis of each graph indicates the number of days of culture after the start of culture. The mouth shows the results of the SM3G1 / CCR4-AF strain, and the garden shows the results of the CH0 / CCR4-AF strain.
- FIG. 3 is a graph showing the binding activity of 11 kinds of anti-CCR4 chimeric antibodies having different ratios of non-fucose-bound sugar chains to soluble small FcaRla.
- the vertical axis shows the binding activity, and the horizontal axis shows the ratio of non-fucose-linked sugar chains.
- ⁇ indicates the binding activity of each anti-CCR4 chimeric antibody, and the solid line in the figure indicates a calibration curve prepared based on the binding activity of each anti-CCR4 chimeric antibody.
- Figure 4 shows the binding activity of the anti-CCR4 chimeric antibody contained in samples collected from serum-free Fued patch cultures of SM3G1 / CCR4-AF strain and CH0 / CCR4-AF strain to soluble human FcaRIIIa. It is.
- the vertical axis indicates the binding activity, and the horizontal axis indicates the number of days after the start of the cultivation of the sample.
- the mouth shows the activity of the sample derived from the SM3G1 / CCR4-AF strain
- the cheat shows the activity of the sample derived from the CH0 / CCR4-AF strain.
- FIG. 5 is a diagram showing the construction of plasmid pBS--.
- FIG. 6 shows the construction of plasmid pKAN-ATM.
- FIG. 7 shows the construction of plasmids pBS-ATIIN135Q and pKAN-ATII1N135Q.
- CH0 / DG44 cells (Proc. Natl. Acad. Sci. USA, 77. 4216 (1980)) were added to IMDM-FBS (10) -HT (1) medium [ ⁇ fetal serum (FBS) (Invitrogen)] was cultured in an IMDM medium (manufactured by Invitrogen) containing 10% HT supplement (manufactured by Invitrogen) in a 75 cm 2 adhesion culture flask (manufactured by Greiner), and grown to just before confluence. . After washing the cells with 5 mL of Dulbecco's PBS (hereinafter referred to as PBS) (Invitrogen), 0.05% trypsin diluted with PBS was used.
- PBS Dulbecco's PBS
- RNeasy Protect Mini Kit manufactured by Qiagen
- RA was prepared according to the attached instruction manual.
- a Super SCRIPT Firs t-Strand synthesis system for RT-PCR manufactured by Impitrogen
- in single-stranded cDNA in a reaction solution of 20 iL from each RNA5 / _tg. was synthesized.
- a 20 L reaction solution containing 0.5 U of single-stranded cDNA from each cell line prepared in (1) of this section as type III [1 XEX Taq Buf fer (Takara Shuzo), 0.2 mM dl TP's, 0.5 units EX Taq polymerase (Takara Shuzo), 0.5 zM synthetic DNA primers of SEQ ID NOS: 11 and 12]
- U-cycle was performed at 94 ° C for 1 minute and at 68 ° C for 2 minutes.
- the expression level of the GDP-mannose 4,6-dehydratase gene of each lectin-resistant CH0 / DG44 cell line obtained in (1) was analyzed.
- the cDNA sequence of GDP-mannose 4,6-dehydratase derived from CH0 cell shown in SEQ ID NO: 1 A 26-mer synthetic DNA primer having the base sequence shown and a 28-mer synthetic DNA primer having the base sequence shown in SEQ ID NO: 14 were prepared.
- a 20 / L reaction solution [1 XEX Taq Buf fer (Takara Shuzo) containing 0.5 zL of single-stranded cDNA derived from each cell line prepared in this section (1) as type I 2 mM dNTP's, 0.5 units of EX Taq polymerase (manufactured by Takara Shuzo Co., Ltd.) and 0.5 / M of the synthetic DNA primers of SEQ ID NOS: 13 and 14] were prepared. After heating at 94 for 5 minutes, a cycle of 94 for 1 minute and 68 at 2 minutes was performed 30 times.
- the CH0 SM strain showed a lectin recognizing the same sugar chain structure as that recognized by LCA, that is, an N-glycoside-linked reduced sugar chain. It also showed resistance to other lectins that recognize a sugar chain structure in which the 6th position of the terminal N-acetyltilcosamine residue and the 1st position of fucose are added by ⁇ -linkage.
- a medium supplemented with endumamelectin (Pisum sat iv abdominal agglutinin; hereinafter, referred to as PSA, manufactured by Vector) having a final concentration of lmg / mL, or a medium with a final concentration of lmg / mL
- PSA endumamelectin
- ML Lec 10r
- Genome analysis of cell lines that do not express the gene for the enzyme that catalyzes the dehydration reaction that converts GDP-mannose to GDP-4-keto and 6-deoxy-GDP-mannose Using CH0 / DG44 cells and the CHO SM strain obtained in Example 1 in IMM-FBS (10)-HT (1) medium until they reach confluence in a T75 flask for adherent cell culture (Grainer) After culturing, genomic DNA is prepared according to a known method [Nucleic Acid Research, 3, 2303, (1976)], and a TE-Rnase buffer (pH 8.0) [10 mmol / l Tris -HCK 1 bandol ol / l EDTA, 200 ig / ml Rnase A] 300 1
- a probe for detecting the FUT8 gene was prepared as follows. First, 10 ⁇ g of the plasmid mfFUT8-pCR2.1 containing the mouse FUT8 cDNA described in Example 11 of W002 / 31140 was dissolved in M buffer (Takara Shuzo) and digested overnight with the restriction enzyme Hindlll (Takara Shuzo).
- reaction solution was replaced with H buffer (Takara Shuzo), and further digested with EcoRI (Takara Shuzo). After completion of the reaction, the reaction solution was subjected to 2% agarose electrophoresis, and then a 156 bp EcoRI-Hindlll fragment containing FUT8 gene exon 2 was purified. 25 ng of the obtained DNA fragment was radiolabeled with 1.75 MBq of [ ⁇ - 32 P] dCTP and Megaprime DNA labeling system, dCTP (Amersham Bioscience).
- Hybridization was performed as follows. First, the above nylon membrane is sealed in a bottle, and 15 mL of hybridization solution [4XSSPE, 5X Denhaldt's solution, 0.5% (w / v) SDS, 0.1 mg / mL salmon sperm DNA] is added, and the mixture is added at 65 ° C. For 3 hours pre-hybridization. Next, the 32 P-labeled probe DNA was denatured by heat, put into a pot, and heated at 65 ° C.
- the nylon membrane was immersed in 50 mL of 2XSSC-0.1% (w / v) SDS and heated at 65 ° C for 15 minutes. After repeating the above washing operation twice, the membrane was immersed in 50 mL of 0.2XSSC-0.1% (w / v) SDS, and heated at 65 T: for 15 minutes. After washing, the nip film was exposed to X-ray film at ⁇ 80 ° C. and developed. After the development, the nylon membrane was boiled in a stripping solution [1% SDS, 0. IX SSC] to release the probe, and hybridization with a different probe was performed again.
- a probe specific to GMD gene exon 5 was prepared as follows. First, based on the known human GMD genomic DNA sequence (NCBI accession number NT-034880), primers (SEQ ID NO: 15 and SEQ ID NO: 16) that specifically bind to exon 5 were designed. This region corresponds to base number 346 to base number 538 of the human GMD cDNA sequence shown in SEQ ID NO: 2.
- a 100 L reaction solution containing 10 ng of the plasmid PAGE249GMD described in Example 15 of TO02 / 31140 [ExTaq buf fer (Takara Shuzo), 0.2 acetyl / L dNTPs, 2.5 ⁇ mol / L Specific primers (SEQ ID NO: 15 and SEQ ID NO: 16)] were prepared and subjected to polymerase chain reaction (PCR).
- the PCR was carried out under the conditions of 30 cycles, in which a reaction consisting of heating at 94 for 5 minutes, 1 minute at 581, 2 minutes at 581, and 3 minutes at 72 ° C was one cycle.
- the anti-CCR4 human chimeric antibody expression plasmid PKMTEX2160 described in TO01 / 64754 was added to the knockout strain CHO SM strain of GDP-mannose 4,6-dehydratase gene and the parent strain CH0 / DG44 cell line obtained in Example 1. introduced, anti-CCR4 human chimeric ⁇ stable production cells on to the anti-CCR4 human chimeric antibody expression base click evening one PKANTEX2160 prepared as follows 4 _ ⁇ 1.
- the culture supernatant was recovered from the wells in which growth was observed, and the expression level of the anti-CCR4 human chimeric antibody in the supernatant was determined as described in this example. It was measured by the ELISA method described in section 2.
- the cells were suspended in IMDM-dFBS (lO) medium containing 50 nM of MTX at a concentration of 1-2 ⁇ 10 5 cells / mL, and (Manufactured by Iwaki Glass Co., Ltd.). After culturing at 37 ° C. for 1 to 2 weeks in a 5% CO 2 incubator, a transformant showing 50 ⁇ resistance was induced.
- the acquired SM3G1 / CCR4 strain is the name of the SM3G1 / CCR4 strain, and the Patented Biological Depositary Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba East 1-chome, Ibaraki Prefecture, Japan, dated September 9, 2003. Deposited as FERM BP-08473 at Address 1 Central 6).
- a goat anti-human IgG (H & L) antibody (American Qualex) was diluted to 1 g / mL with PBS and dispensed at 50 L / well into a 96-well ELISA plate (Grainer One). , 4 overnight for adsorption. After washing with PBS, add PBS containing 1% BSA (hereinafter referred to as 1% BSA-PBS) (manufactured by Wako Pure Chemical Industries, Ltd.) at 100 L / well, and allow to react at room temperature for 1 hour to remain. Active groups were blocked.
- 1% BSA-PBS PBS containing 1% BSA
- the 1% BSA-PBS was discarded, and various dilutions of the antibody purified from the culture of the transformed strain or the culture supernatant were added at 50 / L / well, and the mixture was incubated at room temperature for 1 hour. After the reaction, wash each well with PBS containing Tween20 at a concentration of 0.05% (hereinafter referred to as Tween-PBS) (manufactured by Wako Junjo Co., Ltd.), and then dilute the peroxygen by 2000 times with 1% BSA-PBS. Dase-labeled goat anti-human IgGOKL) antibody solutions (American Qualex) were added as secondary antibody solutions at 50 L / well, and reacted at room temperature for 1 hour.
- Tween-PBS PBS containing Tween20 at a concentration of 0.05%
- ABTS substrate solution [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium (manufactured by Wako Pure Chemical Industries) is added to 1 L 0.1M solution: Dissolve in phosphate buffer (pH 4.2) and add hydrogen peroxide (manufactured by Wako Pure Chemical Industries, Ltd.) at liiL / mL immediately before use. In addition, color was developed, and the absorbance at 415 nm (hereinafter referred to as 0M15) was measured.
- anti-CCR4 human chimeric antibodies produced by each were purified as follows.
- the binding activity of the CH0 / CCR4 antibody and SM3G1 / CCR4 antibody purified in section 3 of this example to human CCR4 antigen was measured as follows.
- Compound 1 (SEQ ID NO: 17) was selected as a human CCR4 extracellular region peptide to which an anti-CCR4 human chimeric antibody can react.
- a conjugate with BSA Bovine Serum Albumin (manufactured by Nacalai Tesque) was prepared by the following method and used as an antigen for use in measuring the activity by the ELISA method.
- the conjugate prepared as above is dispensed into a 96-well EIA plate (manufactured by Grainer Co., Ltd.) at 0.05'Mg / mL, 50 / iL / well, and left at 4 to stand. Adsorbed. After washing with PBS, 1% BSA-PBS was added at 100 L / well, and reacted at room temperature for 1 hour to block the remaining active groups. After washing each well with Tween-PBS, the culture supernatant of the transformed strain was added at 50 AtL / well, and reacted at room temperature for 1 hour.
- ADCC activity of the two purified anti-CCR4 human chimeric antibody preparations obtained in section 3 of this example was determined as follows:
- the measurement was performed as follows using CCR4 / EL4 cells, a mouse thymic cell EL4 cell line that highly expressed human CCR4 described in W001 / 64754 as target cells.
- CCR4 / EL4 cells are centrifuged and suspended to obtain RPMI 1640-FCS (5) medium (RPI 1640 containing 5% FCS). After washing with a medium (GIBCO BRL)), the cells were adjusted to 2X cells / mL with RPMI1640-FCS (5) medium to obtain a target cell solution.
- the plate After the reaction, the plate is centrifuged, and the lactate dehydrogenase (LDH) activity in the supernatant is measured using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) according to the instructions attached to the plate. It was measured. Absorbance data of spontaneous release of target cells is obtained by using only the medium instead of the effector cell solution and antibody solution, and Fefecta-absorbance data of spontaneous release of the cells is obtained by using only the medium instead of the target cell solution and antibody solution was obtained by performing the same operations as above.
- LDH lactate dehydrogenase
- FIG. 1 shows the ADCC activity of the CH0 / CCR4 antibody and SM3G1 / CCR4 antibody on CCR4 / EL4 cells.
- Knockout strain of GDP-mannose 4,6-dehydratase gene Transformant strain obtained from CH0 SM strain SM3G1 / CCR4 strain produced by SM3G1 / CCR4 antibody is approximately 100 times that of CH0 / CCR4 antibody Increased ADCC activity was observed. The results were the same even if the effector cells had different donors.
- Neutral sugar 'amino sugar composition of two purified anti-CCR4 human chimeric antibodies obtained in section 3 of this example The analysis was performed as follows.
- CarboPac PA-1 column and CarboPac PA-1 guard column manufactured by Dionex
- 10-2 MbR sodium oxide, deionized water solution, 500 mM hydroxide sodium-deionized water as washing solution
- analysis was performed using the following elution program.
- composition ratio of each component was calculated from the peak area of the obtained neutral sugar'amino sugar component when the N-acetylglycosamine ratio was set to 4.
- Each transformant is suspended in IMDM-dFBS (10) medium containing cells at a concentration of 500 ⁇ at a cell density of 2-4 10 5 cells / 1 ⁇ , and a 75 cm 2 flask for adherent cells (Grainer And statically cultured at 37 ° C. in a 5% CO 2 incubator.
- the confluent cells were detached with a 0.05% trypsin (Invitrogen) solution, suspended in IM ⁇ -dFBS (10) medium containing MTX at a concentration of 500 nM, and the supernatant was removed by centrifugation. .
- the obtained cells were placed in an EX-CELL302 medium (manufactured by JRH) containing 500 nM of MTX and L-glutamine (manufactured by Invitrogen) at a concentration of 6 (hereinafter referred to as serum-free medium) at 5 ⁇ 10 5 cells / mL. , And 15 mL of the cell suspension was inoculated into a 125 mL Erlenmeyer flask (manufactured by Koning Co., Ltd.). The 5% C0 2 for 4 or more times of the culture vessel was sealed after replacing the air in the flask was vented, 35 ° C, were suspended gyratory at 90 ⁇ 100Rpm.
- SM3G1 / CCM strain adapted to serum-free is referred to as SM3G1 / CCIU-AF
- CH0 / CCR4 strain adapted to serum-free is designated as CH0 / CCR4-AF.
- serum-free feeder culture was performed as follows. ,
- a serum-free medium modified from the previous section was used, and a 20% (w / v) glucose solution was added to a final concentration of 5000 mg / L and used (hereinafter, serum-free). Huedovich culture medium).
- an amino acid prepared at a higher concentration than the usual addition concentration (L-alanine 0.177 g / L, L-arginine monohydrochloride 0.593 g / L, L-asparagine monohydrate 0.177 g / L) L, L-aspartic acid 0.22 g / L, cystine dihydrochloride 0.646 g / L, L-glucamic acid 0.530 g / L, L-glu Tamine 5.84 g / L, glycine 0.22 g / L, L-histidine monohydrochloride dihydrate 0.297 g / L, L-isoleucine 0.742 g / L, L-leucine 0.742 g / L, L-lysine Monohydrochloric acid 1.031 g / L, L-methionine 0.22 g / L, L-phenylalanine 0.466 g / L, L-proline 0.283 g / L
- the SM3G1 / CCR4-AF strain and the CH0 / CCR4-AF strain were suspended in a serum-free Fued batch culture medium at a density of 3 ⁇ 10 5 cells / mL, and 40 mL of the suspension was transferred to a 250 mL Erlenmeyer flask. Ning Co.).
- the 5% C0 2 for 4 or more times of the culture vessel was sealed after replacing the air in the flask was vented at 35, it was 13 days suspension gyratory at 90 ⁇ 100Rpm.
- the results are shown in FIGS.
- the viable cell density (A), cell viability (B), and anti-CCR4 chimeric antibody concentration (C) in the culture supernatant of the SM3G1 / CCR4-AF strain at each time point after the start of culture were all CH0 / CCR4-AF
- the knock-out of the GDP-mannose 4,6-dehydrase gene did not affect cell proliferation or antibody productivity.
- the ratio of sugar chains not linked to a at position 1 of fucose is determined by the soluble human Fc rRI I Ia (hereinafter referred to as shFc r RI I Ia) described in Example 11, paragraph 3 of W003 / 085119.
- shFc r RI I Ia soluble human Fc rRI I Ia
- a human CCR4 extracellular region peptide conjugate was dispensed at a concentration of 1.0 / zg / mL into a 96-well ELISA plate (Grainer) at 50 jL / well, and left overnight at 4 for adsorption. . After washing with PBS, 1% BSA-PBS was added at 100 / iL / well, and reacted at room temperature for 1 hour to block remaining active groups.
- each anti-CCR4 chimeric antibody sample diluted to 5.0 g / inL with 1% BSA-PBS was added at 50 L / well, and reacted at room temperature for 1 hour.
- Fig. 3 shows each anti-CCR4 chimeric antibody prepared in (1) of this section, in which the proportion of sugar chains in which fucose at position 6 is not ⁇ -linked to position 6 of ⁇ -acetyldarcosamine at the reducing end is known. Showed binding activity to shFcrRIIIa. The binding activity to shFcrRIIIa increased in proportion to the proportion of sugar chains in which position 1 of fucose was not ⁇ -linked to position 6 of N-acetyltyldarcosamine at the reducing end.
- FIG. 4 shows the measured binding activity of the anti-CCR4 chimeric antibody to shFcrRIIIa contained in the serum-free Fued batch culture sample collected in section 2 of this example as a measured value of 0D415.
- any of the samples derived from the CH0 / CCR4-AF strain almost no binding activity to shFcrRIIIa was observed.
- the sample derived from SM3G1 / CCR4-AF strain showed strong binding activity to shFcrRIIIa throughout the culture period. .
- Table 3 shows that the 1-position of fucose is not ⁇ -linked to the 6-position of N-acetyldarcosamine at the reducing end of the anti-CCR4 chimeric antibody contained in each culture sample, as determined from the calibration curve in Fig. 3.
- the ratio of sugar chains is shown.
- about 90% or more (> 90%) was shown about the sample which showed the value which exceeded the light absorbency of 2760-1 (90%).
- the proportion of sugar chains in which the 1-position of fucose was not ⁇ -linked to the 6-position of ⁇ ⁇ -acetyldarcosamine at the reducing end was as low as 10 to 11%.
- samples derived from the SM3G1 / CCR4-AF strain showed a high value of 90% or more throughout the culture period.
- Table 4 shows that the first position of fucose binds to the 6th position of the N-acetyldarcosamine at the reducing end of the sugar chain in the total N-glycoside-linked complex type sugar chain, calculated from the monosaccharide composition ratio of each antibody.
- the ratio of sugar chains not used is shown.
- the anti-CCR4 chimera derived from the CH0 / CCR4-AF strain had a fucose-free sugar chain ratio of 15%, whereas the SM3G1 / CCR4-AF-derived anti-CCR4 chimeric antibody derived fucose did not. Since the peak of ⁇ is below the detection limit, the percentage of sugar chains to which fucose is not bound was estimated to be 100%.
- the GDP-mannose 4,6-dehydratase gene knockout strain does not have ⁇ -linked fucose at position 6 at the reducing end of N-acetyldarcosamine even in serum-free fed-batch culture. It was confirmed that an antibody having a sugar chain could be stably produced.
- CH0SM strain which is a knockout strain of the GDP-mannose 4,6-dehydrase gene obtained in Example 1
- N-linkage at position 135 in human antithrompine ⁇ (hereinafter referred to as ⁇ ) and the mature form Stable glycosylation sound
- ATIEN135Q mutant human antithrompine II
- a 20 L reaction solution containing human liver-derived cDNA as a template [PyrobesR DNA Polymerase (Yukarapaio), lOXPyrobest buffer, 0.2 band ol / L dNTP mixture, 0.5 zmol / L
- the above primers (SEQ ID NO: 19 and SEQ ID NO: 20)] was prepared and heated at 94 for 1 minute, and then PCR was performed in a 30 cycle cycle reaction consisting of 98 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 2 minutes. .
- reaction solution was subjected to 1.5% (W / V) agarose gel electrophoresis to confirm a DNA fragment of the ⁇ gene of about 1400 bp, and purified using a QIAquick Gel Extraction Kit (manufactured by QIAGEN).
- the above-obtained ATIE DNA fragment (EcoRI-BamHI) and pBluescriptn KS (+) fragment (EcoRI-BamHI) were subjected to 1.5% (W / V) agarose gel electrophoresis, and about 1400 bp and 3 kb DNA fragments were obtained. Purification was performed using a QIAquick Gel Extraction Kit (manufactured by QIAGEN). Next, a reaction mixture containing 20 ng of the ATIE DNA fragment (EcoRI-BamH I), 80 ng of the pBluescriptn KS (+) fragment (EcoR I-BamH I), and Ligation HigM manufactured by Toyobo Co., Ltd.
- Plasmid DNA is prepared from the transformant using QIAprepR Spin Miniprep it (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Kit v2.0 (QIAGEN) and DNA sequencer ABI PRISM 377 (Aplied Biosystems) was used to analyze the nucleotide sequence. As a result, a plasmid pBS-ATM containing the ⁇ gene sequence shown in FIG. 5 was obtained.
- the plasmid PKANTEX933wg described in item (3) of Example 6 was dissolved in 17.5 L of water.
- 10 units of EcoRI and 2 L of 10XH buffer was added to prepare a 20 iL reaction solution.
- a time digestion reaction was performed.
- phenol / mouth opening form extraction treatment and ethanol precipitation were performed, and the recovered plasmid was dissolved in 17.5 water.
- 10 units of BamHI and 2 L of 10 XK buf fer were added to the solution to prepare a 20 L reaction solution, which was digested at 37 ° C for 16 hours.
- PBS-ATIE fragment (EcoRI-BamHI) and PKANTEX93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (w / v) agarose gel electrophoresis, and the DNA fragments of about 1.4 Kb and 9 Kb, respectively, were subjected to QIAqukk Purification was performed using Gel Extraction Kit (QIAGEN).
- a reaction solution (20 zL) containing 50 ng of the purified pBS-ATIE fragment (EcoRI-BamHI), 30 ng of the purified pKA TEX93 (EcoRI-BamHI) fragment, and Ligation High (manufactured by Toyobo Co., Ltd.) was prepared, and the mixture was prepared at 16 ° C for 16 hours.
- a ligation reaction was performed.
- Escherichia coli DH5a (Toyobo) was transformed by heat shock method.
- Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN), and ⁇ - ⁇ shown in FIG. 6 was obtained.
- Plasmid DNA was prepared from the obtained transformant using QIAprepR Spin Miniprep Kit (QIAGEN), BigDye Terminator Cycle Sequencing Ready Reaction Ki.t v2.0 (QIAGEN) and DNA sequencer ABI The nucleotide sequence was analyzed using PRISM 377 (manufactured by Applied Biosystems). As a result, a plasmid pBS-ATHIN135Q containing the mutant ⁇ gene sequence shown in FIG. 7 was obtained.
- the plasmid pANTEX93 described in item (3) of Example 6 was dissolved in the water of step 17.
- 10 units of 10XH buiier with EcoRI was added to prepare a 20 / L reaction solution, which was digested at 37 for 16 hours.
- the mixture was subjected to phenol / chloroform extraction and ethanol precipitation, and the recovered plasmid was dissolved in 17.5 ⁇ L of water.
- 10 units of BamHI and 2 / L of 10 XK buf fer were added to the solution to prepare a 20 ⁇ l reaction solution, and digestion reaction was carried out at 37 for 16 hours.
- the PBS-ATIEN135Q fragment (EcoRI-BamHI) and ⁇ 93 fragment (EcoRI-BamHI) obtained above were subjected to 1.5% (w / v) agarose gel electrophoresis, and the DNA fragments of about 1.4 Kb and 9 Kb, respectively, were subjected to QIAquick Purification was performed using Gel Extraction Kit (QIAGEN). Then, 50 ng of the purified pBS-AT IE N135Q fragment (EcoRI-BamHI), 30 ng of the purified pKA TEX93 (EcoRI-BamHI) fragment, and Ligation High (manufactured by Toyobo) were used.
- a reaction solution containing 20 ill was prepared, and a ligation reaction was performed at 16 ° C. for 16 hours.
- Escherichia coli DH5 ⁇ ; strain (manufactured by Toyobo) was transformed with the obtained plasmid DNA by the heat shock method.
- Plasmid DNA was prepared from the transformant using QIAprep® Spin Miniprep Kit (manufactured by QIAGEN) to obtain ⁇ - ⁇ N135Q shown in FIG.
- the plasmid pKAN-ATDL PKA -ATIEN135Q prepared in Sections 2 and 4 of this example was introduced into the CHO SM strain prepared in Example 1. These genes were introduced by the following procedure according to the known electoral poration method [Cy to technology, 3, 133 (1990)].
- 30 g of plasmid ⁇ - ⁇ or pKAN-ATIEN135Q was mixed with 20 iL of NEBuffer 3 (manufactured by New Engl and Biolabs) and a reaction solution of 2Q0 / L containing 100 units of restriction enzyme MluI (manufactured by NewEngland Biolabs), At 16 o'clock, linearization was performed by performing a digestion reaction.
- the culture supernatant was removed, and 10% fetal dialysis serum, 50 / xg / mL genyumycin and 50 nM methotrexate IMDM medium supplemented with (MTX) (manufactured by Sigma) was added at 100 L / well.
- MTX methotrexate IMDM medium supplemented with
- the medium was replaced with IMDM medium supplemented with 10% fetal dialysis serum, 50 / g / mL gentamicin and 200 nM MTX in the same manner, and the culture was repeated for every 3 to 4 days for 18 days.
- the formed colonies were transplanted to 24 l elplate (Sigma).
- the medium was changed every 3 to 4 days using IMDM medium supplemented with 10% fetal dialysis serum, 50 ⁇ g / mL gentamicin, and 500 nM MTX, and cultured for 19 days with appropriate expansion.
- a 500 nM MTX resistant strain was obtained.
- the method was in accordance with the attached manual, and the pharmaceutical curve Neuart ® (manufactured by Mitsubishi Pharma Corporation) was used for the calibration curve.
- ⁇ and ATIIIN135Q were expressed at the concentrations of 513 ng / mL and 45.4 ng / mL, respectively, in the culture supernatant of the obtained K-producing strains pKAN-ATIIIl GMDK0 and ATIIIN135Q-producing strain ⁇ - ⁇ 133 ⁇ 46 GMDK0. I confirmed that.
- the pKAN-ATIII l GMDK0 strain and PKAN-ATIIIN135Q6 GMDK0 strain are ⁇ - ⁇ 1 GMDK0 and pKAN-ATIIIN135Q6 (MMO strain name, deposited on August 10, 2004 by the National Institute of Advanced Industrial Science and Technology (AIST)
- FERM BP-10083 and FE Thigh BP-10084 have been deposited at Center 1 (Tsukuba-Higashi 1-chome 1-chome 1-Chuo No. 6 Ibaraki, Japan), and the knockout of GDP-mannose 4, 6-dehydratase gene.
- ⁇ and ATIIIN135Q produced in cells have no fucose modification in their carbohydrate structures and have improved blood stability compared to ⁇ and ATIIIN135Q produced in normal CH0 cells. It was confirmed that a significant difference in pharmacological activity such as binding activity was also observed.
- the present invention it is possible to provide a cell capable of producing a glycoprotein having a high pharmacological activity, a method for producing a glycoprotein using the cell, a glycoprotein produced by the production method, and a use thereof.
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WO2011108502A1 (ja) | 2010-03-02 | 2011-09-09 | 協和発酵キリン株式会社 | 改変抗体組成物 |
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CA2704600C (en) | 1999-04-09 | 2016-10-25 | Kyowa Hakko Kirin Co., Ltd. | A method for producing antibodies with increased adcc activity |
US6946292B2 (en) | 2000-10-06 | 2005-09-20 | Kyowa Hakko Kogyo Co., Ltd. | Cells producing antibody compositions with increased antibody dependent cytotoxic activity |
JPWO2003084569A1 (ja) | 2002-04-09 | 2005-08-11 | 協和醗酵工業株式会社 | 抗体組成物含有医薬 |
US20060223147A1 (en) * | 2004-08-05 | 2006-10-05 | Kyowa Hakko Kogyo Co., Ltd., | Process for producing glycoprotein composition |
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EP1914242A1 (en) | 2006-10-19 | 2008-04-23 | Sanofi-Aventis | Novel anti-CD38 antibodies for the treatment of cancer |
AR063975A1 (es) | 2006-11-28 | 2009-03-04 | Centelion | Fusiones fc con receptor para fgf soluble modificadas con actividad biologica mejorada |
PL2282773T3 (pl) * | 2008-05-02 | 2014-08-29 | Seattle Genetics Inc | Sposoby i kompozycje do wytwarzania przeciwciał i pochodnych przeciwciał o obniżonej fukozylacji rdzeniowej |
AR078470A1 (es) | 2009-10-02 | 2011-11-09 | Sanofi Aventis | Anticuerpos que se unen especificamente al receptor epha2 |
NZ606250A (en) | 2010-08-05 | 2015-04-24 | Seattle Genetics Inc | Methods of inhibition of protein fucosylation in vivo using fucose analogs |
WO2014031875A1 (en) | 2012-08-23 | 2014-02-27 | Seattle Genetics, Inc. | Treatment of sickle cell disease and inflammatory conditions |
WO2019207159A1 (en) | 2018-04-27 | 2019-10-31 | Fondazione Ebri Rita Levi-Montalcini | Antibody directed against a tau-derived neurotoxic peptide and uses thereof |
Citations (3)
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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 |
WO2003085118A1 (fr) * | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production de composition anticorps |
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CA2704600C (en) * | 1999-04-09 | 2016-10-25 | Kyowa Hakko Kirin Co., Ltd. | A method for producing antibodies with increased adcc activity |
US20040093621A1 (en) * | 2001-12-25 | 2004-05-13 | Kyowa Hakko Kogyo Co., Ltd | Antibody composition which specifically binds to CD20 |
-
2004
- 2004-10-08 AU AU2004280066A patent/AU2004280066A1/en not_active Abandoned
- 2004-10-08 CA CA002542130A patent/CA2542130A1/en not_active Abandoned
- 2004-10-08 JP JP2005514670A patent/JPWO2005035741A1/ja active Pending
- 2004-10-08 EP EP04773769A patent/EP1676910A4/en not_active Withdrawn
- 2004-10-08 WO PCT/JP2004/015318 patent/WO2005035741A1/ja not_active Application Discontinuation
Patent Citations (3)
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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 |
WO2003085118A1 (fr) * | 2002-04-09 | 2003-10-16 | Kyowa Hakko Kogyo Co., Ltd. | Procede de production de composition anticorps |
Non-Patent Citations (2)
Title |
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See also references of EP1676910A4 * |
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", BIOTECHNOL. BIOENG., vol. 87, no. 5, 5 September 2004 (2004-09-05), pages 614 - 622, XP002983153 * |
Cited By (7)
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US8048999B2 (en) | 2005-12-13 | 2011-11-01 | Kyoto University | Nuclear reprogramming factor |
US8058065B2 (en) | 2005-12-13 | 2011-11-15 | Kyoto University | Oct3/4, Klf4, c-Myc and Sox2 produce induced pluripotent stem cells |
US8278104B2 (en) | 2005-12-13 | 2012-10-02 | Kyoto University | Induced pluripotent stem cells produced with Oct3/4, Klf4 and Sox2 |
US9714433B2 (en) | 2007-06-15 | 2017-07-25 | Kyoto University | Human pluripotent stem cells induced from undifferentiated stem cells derived from a human postnatal tissue |
US9499797B2 (en) | 2008-05-02 | 2016-11-22 | Kyoto University | Method of making induced pluripotent stem cells |
WO2010018847A1 (ja) | 2008-08-13 | 2010-02-18 | 協和発酵キリン株式会社 | 遺伝子組換えプロテインs組成物 |
WO2011108502A1 (ja) | 2010-03-02 | 2011-09-09 | 協和発酵キリン株式会社 | 改変抗体組成物 |
Also Published As
Publication number | Publication date |
---|---|
EP1676910A4 (en) | 2007-03-14 |
JPWO2005035741A1 (ja) | 2006-12-21 |
EP1676910A1 (en) | 2006-07-05 |
AU2004280066A1 (en) | 2005-04-21 |
CA2542130A1 (en) | 2005-04-21 |
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