WO2007094462A1 - Animal non humain possedant un genome modifie ou sa progeniture - Google Patents

Animal non humain possedant un genome modifie ou sa progeniture Download PDF

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WO2007094462A1
WO2007094462A1 PCT/JP2007/052873 JP2007052873W WO2007094462A1 WO 2007094462 A1 WO2007094462 A1 WO 2007094462A1 JP 2007052873 W JP2007052873 W JP 2007052873W WO 2007094462 A1 WO2007094462 A1 WO 2007094462A1
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seq
protein
amino acid
acid sequence
antibody
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Japanese (ja)
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Naoyuki Taniguchi
Jianguo Gu
Eiji Miyoshi
Naoko Ohnuki
Mitsuo Satoh
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Kyowa Hakko Kogyo Co., Ltd.
Osaka University
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Publication of WO2007094462A1 publication Critical patent/WO2007094462A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • 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]
    • 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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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P37/02—Immunomodulators
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • A61P37/08—Antiallergic agents
    • 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
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • 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
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the activity of an enzyme involved in sugar chain modification in which the 1-position of fucose is a- linked to the 6-position of N-acetylyldarcosamine at the N-glycoside-linked complex sugar chain reducing terminal is tissue-specific.
  • the present invention relates to a non-human animal whose genome has been modified so as to be reduced or deleted, or a progeny thereof, a production method thereof, and a use thereof.
  • sugar chains of glycoproteins are divided into two chains: sugar chains that bind to asparagine (N-glycoside-linked sugar chains) and sugar chains that bind to serine, threonine, etc. (0-glycosyl-linked sugar chains). Broadly divided into types. N-glycoside-linked sugar chains have various structures (see Non-Patent Document 1), but in each case, it is known that they have a basic common core structure represented by the following structural formula (I). Being
  • the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end.
  • N-glycoside-linked glycan high mannose type in which only mannose binds to the non-reducing end of the core structure, or galactose-N-acetylyldarcosamine (hereinafter referred to as Gato GlcNAc) on the non-reducing end of the core structure
  • Gato GlcNAc galactose-N-acetylyldarcosamine
  • a glycoprotein having an N-glycoside-linked sugar chain exists as a heteroglycoprotein composition composed of molecules having a variety of sugar chain structures even if the core protein is the same.
  • the sugar chain structure is thought to be controlled by a glycosyltransferase that synthesizes sugar chains and a glycolytic enzyme that degrades sugar chains.
  • Glycoprotein sugar chains including N-glycoside-linked sugar chains, are the three-dimensional structure of the protein part.
  • Non-Patent Document 2-4 the analysis of congenital disorders of glycosylation (CDG) related to sugar chain abnormalities has reported that the causative gene is a gene required for the synthesis of N-glycoside-linked sugar chains. (See Non-Patent Documents 5 and 6).
  • CDG congenital disorders of glycosylation
  • N-glycoside-linked complex-type sugar chain reducing terminal N-acetylcylcosamine has an enzyme involved in sugar chain modification in which the 1-position of fucose is ⁇ - linked to the 6-position of many sugar chains. Unlike modifying enzymes, so far only a single gene has been found in vivo, and its function has attracted much attention.
  • ⁇ -1,6-fucosyltransferase is the enzyme involved in sugar chain modification in which the 1-position of fucose is ⁇ -linked to the 6-position of ⁇ ⁇ -acetylcylcosamine at the reducing end of the ⁇ -glycoside-linked sugar chain.
  • EC 2.4.1,68 The structure of the -1,6-fucosyltransferase gene (EC 2.4.1,68) was clarified in 1996 (see Non-Patent Documents 10 and 11 and Patent Document 1).
  • Non-patent Document 12 The enzyme activity of 1,6-fucosyltransferase has been confirmed in many organs, and in particular, it has been reported that the enzyme activity is relatively high in the brain and small intestine (Non-patent Document 12). — See 13). It has been pointed out that fucose-modified sugar chains play important physiological functions in retinal formation. Attention has been focused on the regulation of erase expression (see Non-Patent Document 14). Even in blood coagulation, the role of platelet-derived ex-1,6-fucosyltransferase is attracting attention (see Non-Patent Document 15).
  • Non-Patent Documents 16, 16 17
  • ⁇ -1, 6-fucosyltransferase activity increased in some diseases such as liver cancer and cystic fibrosis, and the ratio of the enzyme reaction product increased. Therefore, it is suggested that these diseases are associated with the enzyme (see Non-Patent Documents 18 and 19).
  • Transgenic mice overexpressing ⁇ -1,6-fucosyltransferase have also been produced, and steatosis-like degeneration has been observed in the liver and kidney of the produced transgenic mice (non- (See Patent Document 20).
  • a method for producing a -1,6-fucosyltransferase knockout mouse has also been reported (see Patent Document 2).
  • Patent Document 1 WO92 / 27303
  • Patent Document 2 US2005-0160485
  • Non-Patent Document 1 Tetsuko Takahashi, “Biochemical Experimental Method 23—Glycoprotein Glycan Research Method”, Academic Press Center, 1989, p.1-4
  • Non-Patent Document 2 Current 'Ob-Yon' In 'Immunology (Curr. Opin. Immunol.), 3, 646, 1991
  • Non-Patent Document 3 Glycobiology, 3, 97, 1993
  • Non-Patent Document 4 Biochemical 'Social One' Transaction (Biochem. Soc. Trans.
  • Non-Patent Document 5 Glycobiology, 3, 423, 1993
  • Non-Patent Document 6 Bi-Bien'Journal ⁇ Ob ⁇ Pediatric ⁇ Neurology (Eur. J Paediatr Neurol.), 1, 61, 1997
  • Non-Patent Document 7 Nichiya's Genetix (Nat. Genet.), 28, 73, 2001
  • Non-Patent Document 8 Nichiya's Geneties (Nat. Genet.), 28, 69, 2001
  • Non-Patent Document 9 Biochemical and Biophysical Research Communications (Biochem. Biopnys. Res. Commun.), 72, 909, 197 ⁇
  • Non-Patent Document 10 Journal 'Ob' Biological 'Chemistry (J. Biol. Chem.), 271, 27817, 1996
  • Non-Patent Document 11 Journal 'Ob' Biochem., 121, 626, 1997
  • Non-Patent Document 12 International Journal of Cancer, 72, 1117, 1997
  • Non-patent document 13 Biochim. Biophys. Acta., 14 73, 9, 1999
  • Non-Patent Document 14 Glycobiology, 9, 1171, 1999
  • Non-Patent Document 15 Biochemical Society 'Transaction (Biochem.Soc. Trans.
  • Non-Patent Document 16 Journal 'Ob' Biological Chemistry (J. Biol. Chem.), 277, 26733, 2002)
  • Non-Patent Document 17 Journal 'Ob' Biological Chemistry (J. Biol. Chem.), 278, 3466, 2003
  • Non-Patent Document 18 Hepatology, 13, 683, 1991
  • Non-Patent Document 19 Hepatology, 28, 944, 1998
  • Non-Patent Document 20 Glycobiology, 11, 165, 2001
  • the present invention relates to an enzyme involved in a sugar chain modification in which the 1-position of fucose is ⁇ - linked to the 6-position of N-glycidyl darcosamine at the N-glycoside-linked complex sugar reducing end (hereinafter referred to as “H-; ⁇ 6-).
  • H-; ⁇ 6- The genome is modified so that the activity of ⁇ fucose modifying enzyme '' is abbreviated or deleted in a tissue-specific manner. It is an object to provide a modified non-human animal and a method for using the same.
  • the non-human animal and its progeny of the present invention are considered to be useful for elucidating the physiological role of ⁇ -1,6-fucose modifying enzyme and its relationship with disease states. Development of drugs targeting ⁇ -1,6-fucose modifying enzyme is also useful for the development of glycoprotein drugs.
  • the present invention relates to the following (1) to (20).
  • ⁇ -1,6-fucosyltransferases The following (a) to (i) a non-human animal or a progeny thereof according to (4), which is a protein encoded by DNA that is also selected as a group .
  • ⁇ -1,6-fucosyltransferase is a protein selected from the following (a) to (n) group forces.
  • amino acid sequence represented by SEQ ID NO: 6 one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability, and have an ⁇ -1,6-fucosyltransferase activity.
  • amino acid sequence represented by SEQ ID NO: 9 one or more amino acids are deleted, substituted, inserted and / or added, and the amino acid sequence power is ⁇ -1,6-fucosyltransferase.
  • amino acid sequence represented by SEQ ID NO: 10 one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability, and have ⁇ -1,6-fucosyltransferase activity.
  • (k) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 7 and having ⁇ -1,6-fucosyltransferase activity;
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8 and having ⁇ -1,6-fucosyltransferase activity;
  • a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 10, and having ⁇ -1,6-fucosyltransferase activity.
  • tissue is a tissue from which the following group forces (a) to (l) are selected.
  • Non-human animal powers of the above (1) to (7) which are animals that can be selected for horseshoe, hidge, goats, pigs, horses, mice, rats, -birds, monkeys, and rabbits.
  • tissue or body fluid of the non-human animal or its progeny according to any one of (9) to (12) above is obtained, and the tissue protein is a glycoprotein composition comprising glycoprotein molecules from the body fluid.
  • a method for producing a glycoprotein composition comprising a step of collecting and purifying a product.
  • a glycoprotein composition comprising glycoprotein molecules from the non-human animal according to any one of (9) to (12) above or a progeny thereof obtained by culturing the cell and culturing the cell.
  • a method for producing a glycoprotein composition which comprises collecting and purifying the product.
  • the non-human animal or the progeny thereof according to any one of (9) to (12) is also immortalized by acquiring cells, culturing the immortalized cells, and the culture force glycoprotein molecular force
  • a medicament comprising the glycoprotein composition according to (17) as an active ingredient.
  • a medicament comprising the antibody composition according to (18) above as an active ingredient.
  • the N-glycoside-linked complex type sugar chain reducing terminal N-acetylyldarcosamine A non-human animal whose genome has been modified so that the activity of an enzyme involved in sugar chain modification in which 1-position of fucose is ⁇ - linked at position 6 is reduced or deleted in a tissue-specific manner, a method for producing the same, and a method for using the same.
  • FIG. 1 shows the construction of plasmid mcKOgE2-l.
  • FIG. 2 shows the construction of plasmid pKOScramber901 / ARI.
  • FIG. 3 shows the construction of plasmid mcKOgE2-2.
  • FIG. 4 shows the construction of plasmid mcKOgE2-3.
  • FIG. 5 shows the construction of plasmid mcKOgE2-4.
  • FIG. 6 shows the construction of plasmid mcKOgE2-5.
  • FIG. 7 shows the construction of plasmid mcKOgE2-6.
  • FIG. 8 shows the construction of plasmid mcKOgE2-7.
  • FIG. 9 shows the construction of plasmid mcKOgE2-9.1.
  • FIG. 10 shows the construction of plasmid pKOSelectNeo / ASac.
  • FIG. 11 shows the construction of plasmid pmcKOFUT8Neo.
  • FIG. 12 shows the structure of a tissue-specific targeting vector for mouse Fut8 gene recombination and the Southern blot determination method.
  • FIG. 13 is a view showing a genomic region containing an exon where the translation start codon of the mouse Fut8 gene is located.
  • the non-human animal of the present invention or its progeny is a non-human animal or its progeny whose genome has been altered so that the activity of the ⁇ -1,6-fucose modifying enzyme is reduced or deleted. Any non-human animal or its progeny are also included.
  • Non-human animals include any non-human animals, including, for example, rabbits, hidges, goats, pigs, horses, mice, rats, rabbits, monkeys, and rabbits. Among these non-human animals, non-human animals such as ushi, hidge, mouse, rat, and chicken are preferred.
  • ⁇ -1,6-fucose modifying enzyme is a reaction in which the 6-position of ⁇ -glycidyl glycan-reducing terminal ⁇ -acetyldarcosamine and the 1-position of fucose are a-linked Involved in Any enzyme is included.
  • Specific examples of the ⁇ -1,6-fucose modifying enzyme include ⁇ -1,6-fucosyltransferase.
  • ⁇ -1,6-fucosyltransferase includes the following ( a ), (b), (c), (d), (, (£), (g), (h), (i) or Is the protein encoded by DNA of (j), or (k), (1), (m), (n), (o), (p), (q), (r), (s), ( t), (u), (v), (w), (x) or (y) protein.
  • DN ⁇ ⁇ ⁇ that encodes a protein that has been orbridized under stringent conditions with DNA having the base sequence ability represented by D SEQ ID NO: 1 and has ⁇ -1,6-fucosyltransferase activity
  • amino acid sequence represented by SEQ ID NO: 7 one or more amino acids have been deleted, substituted, inserted and / or added, and the amino acid sequence has an ability to exhibit ⁇ -1,6-fucosyltransferase activity.
  • amino acid sequence represented by SEQ ID NO: 8 one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability, and have an ⁇ -1,6-fucosyltransferase activity.
  • amino acid sequence represented by SEQ ID NO: 9 one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability, and ⁇ -1,6-fucosyltransferase activity.
  • amino acid sequence represented by SEQ ID NO: 10 one or more amino acids are deleted, substituted, inserted and / or added, and have an amino acid sequence ability, and ⁇ -1,6-fucosyltransferase activity.
  • (V) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 7 and having ⁇ -1,6-fucosyltransferase activity;
  • (w) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 8 and having ⁇ -1,6-fucosyltransferase activity;
  • (X) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 9 and having ⁇ -1,6-fucosyltransferase activity;
  • the DNA that hybridizes under stringent conditions is, for example, DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, 3, 4 or 5, or the DNA thereof.
  • DNA such as DNA having the base sequence represented by SEQ ID NO: 1, 2, 3, 4 or 5, or the DNA thereof
  • DNA that can be hybridized when calculated using BLAST, FASTA or the like, the base sequence represented by SEQ ID NO: 1, 2, 3, 4 or 5 is at least 70% or more, preferably 80% or more. % Or more, more preferably 90% or more, still more preferably 95% or more, particularly preferably 98% or more, and most preferably 99% or more.
  • the amino acid sequence represented by SEQ ID NO: 6, 7, 8, 9 or 10 has an amino acid sequence ability in which one or more amino acids are deleted, substituted, inserted and Z or added.
  • Proteins with ⁇ -1,6-fucosyltransferase activity are molecular and 'cloning 3rd edition, current' protocols 'in' molecular ⁇ ⁇ Biology, Nucleic Acids Research, 10, 6487 (1982) , Proc. Natl. Acad. Sci., USA. 79, 6409 (1982), Gene, 3 4, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad.
  • the number of amino acids to be deleted, substituted, inserted, and Z or added is 1 or more, and the number is not particularly limited.
  • the number can be substituted or added, and is, for example, 1 to several tens, preferably 1 to 20, more preferably 1 to 10, and further preferably 1 to 5.
  • one or more amino acids are deleted, substituted or added means that there is a deletion, substitution or attachment of one or more amino acids at any position in the same sequence, Amino acids that are substituted or added, even if deletions, substitutions or additions occur simultaneously, may be natural or non-natural.
  • Natural amino acids include L-alanin, L-asparagine, L-aspartic acid, L-arginine, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-sip Icin, L-lysine, L-methionine, L-phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-parin, L-cysteine and the like.
  • amino acids that can be substituted with each other are shown below. Amino acids contained in the same group can be substituted for each other.
  • Group A Leucine, Isoleucine, Norleucine, Norin, Norpaline, Alanine, 2-Aminobutanoic acid, Methionine, 0-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexinolealanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-amino Adipic acid, 2-aminosuberic acid
  • Group D lysine, arginine, ornithine, 2,4-dianaminobutanoic acid, 2,3-dianaminopropionic acid
  • Group E proline, 3-hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • the protein having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 6, 7, 8, 9 or 10 and having ⁇ -1,6-fucosyltransferase activity And at least 80% or more, preferably 85% or more, and a protein having the amino acid sequence set forth in SEQ ID NO: 6, 7, 8, 9 or 10 when calculated using analysis software such as BLAST and FASTA It means that the protein is preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more, and most preferably 99% or more.
  • non-human animal or its progeny of the present invention examples include a non-human animal or its progeny whose genome has been modified so that the above enzyme activity is reduced or deleted in a tissue-specific manner.
  • the genome has been modified so that the activity of ⁇ -1,6-fucose modifying enzyme is decreased or lost in a tissue-specific manner, for example, bone marrow, lymphocyte thread and tissue (splenocytes or sputum cells).
  • Cells cranial nerve tissue, mammary gland tissue, reproductive tissue, and lung tissue, or the like, or a mutation is introduced into the expression regulatory region of the gene so that the expression of the enzyme is specifically reduced or deleted. It means introducing a mutation into the amino acid sequence of the gene so as to reduce or delete the function of the enzyme.
  • the enzyme activity is specifically decreased or deleted in tissues such as bone marrow, lymphocyte tissue (splenocytes or sputum cells), cranial nerve tissue, mammary gland tissue, reproductive tissue, and lung tissue. Specifically, it means that the enzyme activity is deleted or reduced.
  • introducing a mutation means that the base sequence on the genome is modified by deletion, substitution, insertion, and insertion or addition, and the modified genomic gene. Completely suppress the expression or function of the protein encoded by its genomic gene It is said to be knocked out.
  • a specific example of knocking out a genomic gene is one in which all or part of the target gene has been deleted from the genome.
  • any method can be used as long as the target genomic gene can be modified. Specifically, the homozygote inserted on the chromosome targeted by the target vector for gene disruption is crossed with a tissue-specific non-human animal that expresses the recombinase enzyme.
  • a method for producing a specifically destroyed solid, and a clone individual produced using the nucleus of a cell such as an embryonic stem cell or somatic cell that has undergone targeted genetic modification and a tissue-specific recombinase enzyme examples include a method of producing a solid in which a target gene is destroyed in a tissue-specific manner by mating with an expressed transgenic non-human animal.
  • the N-glycosidic complex-type sugar chain reducing terminal N-acetylcylcosamine is located at the 6th position of fucose (the activity of the enzyme involved in X-linked sugar chain modification is reduced or lost in a tissue-specific manner).
  • a non-human animal or its progeny whose genome has been modified has a higher activity of the enzyme than a non-human animal or its progeny whose genome has been modified so that the enzyme activity is reduced or deleted in a non-specific manner. Since the effect of the drop or deletion on the individual can be limited or reduced, the period in which surviving individuals can be used for observation can be extended.
  • the non-human animal and its progeny of the present invention can be produced by using a method of destroying a gene in a tissue-specific manner by targeting the gene of ⁇ -1,6-fucose modifying enzyme.
  • a-1,6-fucose modifying enzyme include ⁇ -1,6-fucosyltransferase.
  • the method for disrupting a gene in a tissue-specific manner includes any method as long as it can disrupt a gene of a target enzyme in a tissue-specific manner.
  • a method of disrupting a gene in a tissue-specific manner using a homologous recombination method can be mentioned. These will be described in detail below.
  • the non-human animal and its progeny of the present invention target ⁇ -1,6-fucose modifying enzyme gene, perform target-specific gene disruption of the target gene on the chromosome using homologous recombination method, It can be produced by modifying this enzyme gene.
  • tissue-specific gene disruption methods using homologous recombination methods include Cre / loxP system (Cell, 73, 1155 (1993); Science, 265, 103 (1994)) or FLP / FRT system (Science). , 25 9, 984 (1993); Catalog No. 218405 (Stratagene)).
  • the Cre / loxP system is based on the base sequence of the genomic DNA! /, Using the FLP / FRT system, the target gene to be modified (for example, the structural gene of ⁇ -1,6-fucose modifying enzyme, or the promoter gene A target vector for homologous recombination.
  • the target gene to be modified for example, the structural gene of ⁇ -1,6-fucose modifying enzyme, or the promoter gene A target vector for homologous recombination.
  • the prepared target vector is introduced into embryonic stem cells, and cells that have undergone homologous recombination between the target gene and the target vector are selected.
  • the selected embryonic stem cells are introduced into a fertilized egg according to a known injection chimera method or assembly chimera method, and the introduced fertilized egg is transplanted into the oviduct or uterus of a pseudopregnant female mouse to select a germline chimera.
  • Target vector introduced from offspring bred after mating selected germline chimeras An individual having a chromosome inserted by homologous recombination with a gene region on the genome encoding 1S a -1,6-fucose modifying enzyme is selected.
  • the introduced target vector in both homologous chromosomes from the offspring born will undergo homologous recombination with the gene region on the genome that encodes the a-1,6-fucose modifying enzyme.
  • the homozygote having the inserted chromosome is selected.
  • the obtained homozygote was crossed with a transgenic individual expressing Cre recombinase or FLP recombinase in a tissue-specific manner, and the target gene was tissue-specific compared to the tissue expressing these recombinases. Gains an individual that is destroyed by destruction.
  • tissue-specific homozygote having the target gene destroyed is crossed, and a tissue-specific target gene-disrupted homozygote showing the same expression system as the parent is obtained from the progeny.
  • Non-human animals and their offspring can be produced. These are described in detail below.
  • Examples of the method for obtaining cDNA of ⁇ -1,6-fucose modifying enzyme include the methods described below.
  • the prepared total RNA or mRNA force also creates a cDNA library.
  • ⁇ -1,6-fucose modifying enzyme for example, human amino acid sequence
  • a degenerative primer is prepared.
  • a gene fragment encoding -1,6-fucose modifying enzyme is obtained.
  • a cDNA library can be screened to obtain cDNA encoding an ⁇ -1,6-fucose modifying enzyme.
  • the animal cell mRNA may be a commercially available product (for example, Clontech) or may be prepared as follows.
  • Cellular power is also measured by methods such as guazine thiocyanate-cesium trifluoroacetate method [Methods in En zymology, 154, 3 (1987)], guanidine acid thiocyanate '. Examples include phenol's black mouth form (AGPC) method [Analytical 'Analytical Biochemistry, 162, 156 (19 87); experimental medicine, 9, 1937 (1991)].
  • AGPC phenol's black mouth form
  • RNA can be prepared by using a kit such as Fast Track mRNA Isolation Kit (Invitrogen) or Quick Prep mRNA Purification Kit (Pharmacia).
  • a cDNA library is prepared from the mRNA of the prepared animal cells.
  • cDNA library preparation methods include those described in Molecular 'Crowing 2nd Edition, Current' Protocols 'in' Molecular ⁇ Biology, A Laboratory Manual, 2nd Ed. (1989), etc., or commercially available. Examples include kits such as Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Life Technologies) and ZAP-cDNA Synthesis Kit (STRATAGE NE).
  • any phage vector, plasmid vector, etc. can be used as long as it can autonomously replicate in Escherichia coli K12.
  • ZAP Express [STRATAGENE, Strategies, 5, 58 (1992)]
  • pBluescript II SK (+) [Nucleic Acids Research, ⁇ 7, 9494 (1989) ]
  • Lambda ZAP II (STRATAGENE), ⁇ gtl0, ⁇ gtl l [DNA cloning, A Practic al Approach,!, 49 (1985)]
  • any microorganism can be used, preferably Escherichia coli.
  • Science 778 (1 983) 1, Escherichia coli Y1090
  • Science, 222, 778 (1983) 1, Escherichia c 2U NM522
  • Journal 'Ob' Molecular ⁇ Biology (J. Mol. Biol.) 166, 1 (1983) 1, Escherichia coli K802 [Journal 'Ob' Molecular ⁇ ⁇ Biology (J. Mol. Biol.), 16. 118 (1966)]
  • Escherichia coli JM105 [Gene ( Gene), 38, 275 (1985)] Used.
  • this cDNA library can be used for subsequent analysis as it is, an oligo developed by Kanno et al. Has been developed in order to reduce the proportion of incomplete cDNA and to obtain full-length cDNA as efficiently as possible.
  • Cap method [Gene, 138, 171 (1994); Gene, 200, 149 (1997); protein nucleic acid enzyme, 41, 603 (1996); experimental medicine, U, 2491 (1993); cDNA cloning (Yodosha) (1996); cDNA library prepared using gene library preparation method (Yodosha) (1994)] may be used for the following analysis.
  • the obtained gene fragment is a DNA encoding an a-1,6-fucose modifying enzyme.
  • a nucleotide sequence analyzer such as The National Academy of Science (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)] or ABIPRISM377 DNA Sequencer (PE Biosystems) This can be confirmed by analysis.
  • a cDNA or cDNA library synthesized from mRNA contained in mouse cells to be modified is paired with a colony hybridization or plaque hybridization (Molecula 1 ' By performing Cloning 2nd edition), ⁇ -1,6-fucose modifying enzyme DNA can be obtained.
  • a cDNA library synthesized from mRNA contained in animal cells that has been modified using the primers used to obtain the gene fragment encoding the ⁇ -1,6-fucose modifying enzyme.
  • the ⁇ -1,6-fucose-modifying enzyme DNA can also be obtained by screening using the PCR method with the ⁇ as a type.
  • the base sequence of the DNA encoding the obtained a-1,6-fucose modifying enzyme is usually Nucleotide sequence analysis methods used, such as the Sidi et al. Dideoxy method (Proc. Natl. Acad. Sci. USA), 74, 5463 (1977)] or by using a base sequence analyzer such as ABIPRISM377 DNA Sequencer (manufactured by PE Biosystems) to determine the base sequence of the DNA.
  • nucleotide sequence databases such as GenBank, EMBL, and DDBJ, it is also possible to determine genes encoding ⁇ -1,6-fucose modifying enzymes among the genes in the databases.
  • Examples of the base sequence of the gene encoding the a-1,6-fucose modifying enzyme obtained by the above method include the base sequence described in SEQ ID NO: 1, 2, 3, 4 or 5. .
  • Examples of the method for preparing genomic DNA of ⁇ -1,6-fucose modifying enzyme include the methods described below.
  • genomic DNA of ⁇ -1,6-fucosase can be isolated by using genomic DNA library screening system (Genome Systems) or Universal GenomeWalker TM Kits (CLONTECH). .
  • the base sequence of the genomic DNA of the a-1,6-fucose modifying enzyme obtained by the above method includes the cDNA sequence of the a-1,6-fucose modifying enzyme obtained by the above method. Confirm with! /.
  • Target vectors for homologous replacement of target genes to be modified using the Cre / loxP system or FLP / FRT system are Gene Targeting, A Practical Approach, IRL. It can be prepared by the method described in Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells (Yodosha) (1995).
  • the target vector can be a replacement type, an insertion type, a gene trap type, or a misalignment.
  • embryonic stem cells include in vitro cells established from inner cell masses constituting early embryos such as sushi, hidge, goat, pig, horse, mouse, rat, chicken, monkey and rabbit. Examples thereof include cells having pluripotency that can be cultivated in the mouth and can be differentiated into all the cells constituting the living body. Specifically, ES cells derived from the inner cell mass constituting the early embryo before implantation, EG cells derived from primordial germ cells, and so on. Prepared according to the method described in Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Biomanual Series 8 Gene Targeting, Production of Mutant Mice Using ES Cells (Yodosha) (1995) be able to.
  • any method for introducing a target vector into embryonic stem cells any method can be used as long as it introduces DNA into animal cells.
  • the electoral position method [Cytotechnology, 3, 133 (1990 )]
  • Calcium phosphate method [JP-A-2-22707 5]
  • lipofusion method [Procedures of the National Academy of Sciences ( proc . Natl. Acad. Sci. USA), 84, 7413 (1987)]
  • injection method [Mapping, Mouse Embryo 2nd edition]
  • method using particle gun pattern No. 2606856, patent No.
  • a recombinant inserted randomly into the chromosome other than the homologous recombination that cultivates the embryonic stem cells into which the vector has been introduced and selects the growing strain Since it is integrated into the chromosome and expressed because it cannot grow due to the toxicity of DT, negative selection for selecting homologous recombinants that do not contain the DT gene can be performed.
  • Southern hybridization method Molecular ⁇ Cloning 2nd edition
  • genomic DNA and PCR method [PCR] 'Protocols (PCR Protocols), Academic Press (1990)].
  • chimera chimera method When incorporating embryonic stem cells into a fertilized egg using the chimera chimera method, it is generally preferable to use a fertilized egg at the developmental stage prior to the 8-cell stage. When embryonic stem cells are incorporated into a fertilized egg using an injection chimera method, it is generally preferable to use a fertilized egg from the 8 cell stage to the blastocyst development stage.
  • a fertilized egg When a fertilized egg is transplanted into a female mouse, the fertilized egg obtained in a pseudopregnant female mouse in which fertilization ability has been induced is artificially transplanted by mating with a non-human mammal ligated with a vas deferens.
  • Pseudopregnant female mice that prefer the method of implantation can also be obtained by natural mating, but luteinizing hormone-releasing hormone (hereinafter abbreviated as LHRH) is to be bred to male mice after administration of the analog.
  • LHRH luteinizing hormone-releasing hormone
  • LHRH analogues examples include [3,5-DiKTyr5] -LHRH, [Gln8] -LHRH, [D-Ala6] -LHRH, des-GlylO- [D-His (Bzl) 6] -LHRH ethylamide, etc. can give.
  • the selected germline chimera was crossed and inserted from the offspring by homologous recombination with the gene region on the genome encoding the introduced target vector force a-1,6-fucose modifying enzyme. Select individuals with chromosomes.
  • the introduced target vector in both homologous chromosomes from the offspring born will undergo homologous recombination with the gene region on the genome that encodes the a-1,6-fucose modifying enzyme.
  • the homozygote having the inserted chromosome is selected. Tissue-specific Cre recombinase or FLP recombinase is expressed to produce transgenic individuals
  • Trans transgenic individuals expressing Cre recombinase or FLP recombinase in a tissue-specific manner include known tissue-specific promoters [Nat Rev Genet, 2 (10), 743 (2001); J Endocrinol, 171 (1) 1 (2001); Methods Mol Biol, Cliff, 175 (2002); Dev Cell, 6 (1), 7 (2004); Nutr Rev, 62, 243 (2004)].
  • Transgenic animals that express nuclease or FLP recombinase are also included. Specific examples include Cre recombinase or FLP recombinase specifically expressed in bone marrow tissue, lymphocyte tissue (B cell or T cell), cranial nerve tissue, mammary gland tissue, reproductive tissue, lung tissue, etc. Transgenic animals.
  • transgenic animals include, for example, Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993), Novel Manual Series 8 Gene Targeting, Production of Mutated Mice Using ES Cells (Yodo Corporation) ( 1995) and the like, and can be produced using a known transgenic animal production method.
  • the Jackson Laboratory 600 Main Street, Bar Harbor, ME 04609-150 (0, USA) and other public institutions are also available.
  • the resulting homozygote is mated with a transgenic individual expressing Cre recombinase or FLP recombinase in a tissue-specific manner, and the target gene is destroyed in a tissue-specific manner by crossing the tissue where these recombinases are expressed. Get an individual who is being struck.
  • Non-human animals and their progeny of the present invention can be produced.
  • Non-human animals and their progeny of the present invention are disclosed in known literature (Wilmut et al .; Nature, 385, 810 (1997), Cibelli et al .; Science, 280, 1256 (1998), Akira Iriya et al .; Protein Nucleic Acid Enzyme, 44, 892 (
  • the desired alteration is introduced into the gene of the ⁇ -1,6-fucose modifying enzyme on the chromosome of any cell of the non-human animal.
  • the nucleus of the obtained cell is initialized (an operation to return the nucleus to a state where it can be repeated again).
  • Development is initiated by injecting the nucleus of the reprogrammed cells into an enucleated non-fertilized egg of a non-human animal.
  • a heterozygote in which a desired modification is introduced into a gene of a-1,6-fucose-modifying enzyme by artificially transplanting and implanting an egg that has started development into a female non-human animal obtain.
  • a clone individual that is a homozygote is obtained.
  • the resulting homozygous cloned individuals were tissue-specifically Cre recombinase-expressing FLP recombinase and crossed with transgenic individuals, and these recombinases were expressed.
  • the target gene is specifically destroyed and an individual is obtained.
  • tissue-specific target gene disruption homozygotes By crossing the obtained tissue-specific target gene disruption homozygotes with each other and obtaining a tissue-specific target gene disruption homozygote showing the same expression system as its parent, Human animals and their offspring can be produced.
  • a plurality of methods are already known as a method for initializing the cell nucleus.
  • the following method is known.
  • the medium is 5 to 30%, preferably 10% from a medium containing fetal calf serum (eg, M2 medium) for 3 to 10 days.
  • a medium containing fetal calf serum eg, M2 medium
  • the cell cycle is changed to a resting state (GO phase or G1 phase) by culturing in an oligotrophic medium containing 0 to 1%, preferably 0.5% fetal calf serum, preferably for 5 days.
  • a resting state GO phase or G1 phase
  • initialization is performed by injecting the nucleus of the cell providing the nucleus into a slow-nucleated unfertilized egg of the same kind of non-human animal and culturing for several hours, preferably about 1 to 6 hours. Can do. This method is suitable when the non-human animal is, for example, a mouse.
  • Initialized nuclei can begin to develop in enucleated unfertilized eggs.
  • a method for starting development in an unfertilized egg that has been enucleated from an initialized nucleus a plurality of known methods are known.
  • Oocytes are activated by transplanting the reprogrammed nuclei into the resting state (GO phase or G1 phase) of the cell cycle into unfertilized eggs of the same kind of non-human animals that have been gradually nucleated, such as by electrofusion. Generation can be started. This method is suitable when the non-human animal is, for example, a hidge, goat or ushi.
  • Nuclei initialized by injecting nuclei into slow-nucleated unfertilized eggs of the same kind of non-human animals are transplanted again into un-fertilized eggs of the same kind of non-human animals that have been slowly nucleated by a method using a micromanipulator.
  • an egg active substance e.g., strontium
  • a cell division inhibitor e.g., cytochalasin B
  • Examples of a method for artificially transplanting and implanting an egg that has started to develop into a female non-human animal include the method described in 1. (1) above.
  • the genome is altered so that the activity of ⁇ -1,6-fucose modifying enzyme is reduced or deleted in a tissue-specific manner. 2) the physiological role of the enzyme in the process from development to adulthood, and 3) the physiological role of the enzyme in adulthood.
  • test substance is administered to the non-human animal of the present invention or its offspring, and compared with animals not administered with the test substance, various physical properties such as blood pressure, respiratory rate, and body weight of the animal are compared.
  • Pharmacological evaluation of the test substance can be performed by examining pharmacological actions such as measurement of parameters, appearance and behavior observation, and histopathological examination.
  • test substance is administered to the pathological model animal, blood pressure, respiratory rate, body weight, etc. ⁇ -1,6 by measuring various physical parameters, pathologic appearance, appearance, behavioral observation, histopathological examination, etc. in comparison with the model animal without administration of the test substance.
  • the test substance in humans with reduced activity of fucose modifying enzyme Can be evaluated for pharmacological effects such as efficacy and side effects on the disease. Based on this evaluation, a substance preferable as a therapeutic agent for the disease can be selected.
  • Diseases induced in the non-human animal and its progeny of the present invention include heart diseases (for example, acute heart failure, chronic heart failure, myocarditis, etc.), respiratory diseases, joint diseases (for example, rheumatoid arthritis, Osteoarthritis), kidney disease (eg, renal failure, glomerulonephritis, IgA nephropathy), arteriosclerosis, psoriasis, hyperlipidemia, allergic disease (eg, asthma, allergic rhinitis, atopy) Dermatitis, etc.), bone diseases (eg, osteoporosis, rickets, osteomalacia, hypocalcemia), blood diseases, cerebrovascular injury, traumatic brain injury, infection, dementia, cancer, diabetes Liver diseases, skin diseases, neurodegenerative diseases and chronic inflammatory diseases.
  • heart diseases for example, acute heart failure, chronic heart failure, myocarditis, etc.
  • respiratory diseases for example, rheumatoid arthritis, Osteoarthritis
  • joint diseases
  • the non-human animal of the present invention and its progeny are brought into contact with various cells obtained from the test substance, and various kinds of cells such as an increase in intracellular Ca 2+ concentration are compared with cells in the absence of the test substance.
  • the pharmacological evaluation of the test substance on the cells can be performed by examining pharmacological actions including changes in cell response and changes in cell morphology.
  • various types of cells can be obtained by inducing embryonic stem cells from which the non-human animal of the present invention and its progeny can be obtained. Differentiation can be induced by transplanting embryonic stem cells subcutaneously to allogeneic strains of animals by inducing teratomas mixed with various tissues (Meruplating 'Mouse' Embrio) 2nd edition), by culturing in vitro under appropriate conditions, endoderm cells, ectoderm cells, mesoderm cells, blood cells, endothelial cells, chondrocytes, skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons, glia Examples thereof include a method of inducing differentiation into cells, epithelial cells, melanocytes, and keratinocytes (Repro d.
  • the activity of ⁇ -1,6-fucose modifying enzyme is made tissue-specific by the method described in 1. above. It is possible to obtain a genetically modified animal in which the genome is modified so as to decrease, and the gene on the chromosome is further modified.
  • a knockout mouse in which a gene known to cause a specific disease state by disrupting its function is deleted using the homologous recombination technique described in 1. above, Transgenic mice in which a dominant negative gene that inhibits the function of the protein encoded by the gene is introduced and expressed are useful as animal models of pathological conditions.
  • the activity of ⁇ -1,6-fucose modifying enzyme is decreased or lacked in a tissue-specific manner. It is possible to obtain genetically modified animals whose genome has been modified so as to be lost and which exhibit a certain expression system (for example, symptoms similar to human pathologies). If the animal to be mated is a human disease model animal, the genome is modified so that the activity of a-1,6-fucose modifying enzyme is decreased or deleted in a tissue-specific manner, and the symptoms are similar to those of human pathology. The disease model animal shown as a phenotype is obtained.
  • in vitro fertilization can be used as a method of mating.
  • a disease model animal any disease model animal can be used regardless of a disease, congenital or acquired.
  • the acquired animal model of pathological condition is “Municular disease model mouse” [Molecular Medicine, ai special issue, Nakayama Shoten (1994)], “Illustration: Pathological animal model for pharmacology” [Nishimura Shoten (1984)], "arthritis model animals” (Dental and dentistry (1985)), “neurological and muscle disease model animals” (Dental and dentistry (1982)), "active acids It can be prepared by the method described in “Toward Bedside” [Society Publishing Center (1992)].
  • the test substance is administered to the genetically modified disease model animal obtained by the method described in (4) and (5) above, and various physical parameters such as blood pressure, respiratory rate, body weight, etc. of the disease model animal are measured.
  • various physical parameters such as blood pressure, respiratory rate, body weight, etc. of the disease model animal are measured.
  • data such as values, pathological conditions, appearance, behavioral observation, histopathological examination, etc.
  • Pharmacological evaluation such as side effects can be performed.
  • a substance preferable as a therapeutic agent for the disease state can be selected.
  • the genome of the non-human animal and its progeny of the present invention has been modified so that the activity of the ⁇ -1,6-fucose modified enzyme is reduced or deleted in a specific tissue, the Glycoproteins produced in this way have reduced or lacked glycosylation by -1,6-fucose modifying enzymes.
  • glycoproteins with reduced or deleted glycosylation by a-1,6-fucose modifying enzyme interact with proteins necessary for changes in hemodynamics and distribution in the living body and expression of pharmacological activity. Is changing and is useful as a medicine.
  • a gene encoding a glycoprotein molecule is introduced into the non-human animal and its progeny of the present invention, and a glycoprotein composition having glycoprotein molecular strength is produced using the animal and its progeny.
  • the sugar chain is linked to the sugar protein (N-glycoside-linked sugar chain) and to the serine or threonine (0-glycoside bond), depending on the binding mode with the glycoprotein moiety.
  • Sugar chains N-glycoside-linked sugar chain
  • N-glycoside-linked sugar chains have various structures, but even in the case of deviation, they have a common core structure represented by the above structural formula (I).
  • Structural Formula (I) the end of the sugar chain that binds to asparagine is called the reducing end, and the opposite side is called the non-reducing end.
  • the N-glycoside-linked sugar chain is a high mannose-type sugar chain in which only mannose is bonded to the non-reducing end of the core structure, and galactose-N-acetylyldarcosamine (hereinafter referred to as Ga ⁇ GlcNac) on the non-reducing end of the core structure
  • Ga ⁇ GlcNac galactose-N-acetylyldarcosamine
  • Table The ) In parallel with one or more branches, and also has a structure such as sialic acid or bisecting N-acetylyldarcosamine at the non-reducing end of Gato GlcNac (also called complex type). Examples include hybrid sugar chains having both high-mannose and complex branches at the non-reducing end of the chain and core structure.
  • N-acetylylgalatatosamine As the 0-glycoside-linked sugar chain, the reducing end of N-acetylylgalatatosamine is bonded to the hydroxyl group of serine or threonine, and further, galactose, N-acetylyldarcosamine, N-acetylgalato
  • samines include sugar chains to which sialic acid is bonded, sugar chains in which xylose is ⁇ -bonded to the hydroxyl group of serine, and sugar chains in which galactose is ⁇ -bonded to the hydroxyl group of hydroxylidine.
  • a sugar chain in which xylose is bonded to the hydroxyl group of serine in a / 3 bond usually has a plurality of sugars bonded to the 4-position of the xylose, and a linear polysaccharide consisting of two sugars is bonded to the end of the bonded sugar. is doing.
  • An example of a substance having such a sugar chain structure is cartilage proteodarican.
  • Examples of the substance having a sugar chain structure in which galactose is ⁇ -bonded to a hydroxyl group of hydroxylysine include collagen.
  • sugar constituting the sugar chain examples include ⁇ -acetyl darcosamine, ⁇ -acetyl galatatosamine, mannose, galactose, fucose, sialic acid, xylose, arabinose, and the like. You can combine them in the order
  • the glycoprotein composition refers to a composition having a glycoprotein molecular force having a ⁇ -glycoside-linked sugar chain or a 0-glycoside-linked sugar chain.
  • glycoproteins include antibodies, erythropoietin, thrombopoietin, tissue-type plasminogen activator, prolokinase, thrombomodulin, antithrombin III, protein, blood coagulation factor VII, blood coagulation Factor VIII, blood clotting factor IX, blood clotting factor X, gonadotropin, thyroid stimulating hormone, epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor, activin, osteogenic factor, stem cell factor (SCF), interferon ⁇ , interferon 13, interferon ⁇ , interleukin 2, interleukin 6, interleukin 10, interleukin 11, soluble interleukin 4 receptor, tumor necrosis factor at, DNaseI, galactosidase, oc darcosidase, darco Cerebrosidase A more specific example of a glycoprotein whose physiological activity is significantly increased by having a sugar chain structure without fuco
  • the antibody composition is a composition having an N-glycoside-bonded complex type sugar chain in the Fc region and also having an antibody molecular force.
  • a method for producing a glycoprotein composition using the non-human animal of the present invention and its progeny will be described with reference to production of an antibody composition.
  • the antibody composition is Molecura Ira 'Cloning 3rd Edition, Current'Protocols' In 'Mollyph ⁇ ' Noiolon 1 ⁇ , Antioodies, A Laboratory manual, Cold bpnng Haroor La boratory , 1988 (hereinafter abbreviated as anti-bodies), Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press, 1993 (hereinafter abbreviated as monoclonal anti-bodies), Antibody Engineering, A Practical Approach, IRL Press at Oxford Using the method described in University Press, 1996 (hereinafter abbreviated as “antibody engineering”), for example, it can be expressed and obtained in the non-human animal of the present invention as follows.
  • cDNA of the antibody molecule is prepared.
  • a DNA fragment having 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 the downstream of the promoter of an appropriate expression vector.
  • the obtained transformed individual and the non-human animal of the same kind of the present invention produced in the above 1. were mated, and ⁇ -1, 6-fucose was tissue-specifically selected from the offsprings in the same manner as the non-human animal of the present invention.
  • An individual in which the modifying enzyme gene is destroyed and the introduced antibody gene is expressed can be obtained.
  • non-human animal into which a vector expressing an antibody molecule is introduced a non-human animal into which an enzyme related to modification of an N-glycoside-linked sugar chain that binds to the Fc region of the antibody molecule is introduced using a genetic engineering technique. Animals can also be used.
  • Non-human animals producing human antibodies are known literature [Nature Biotech., 14, 845 (1996); Nature Genetics, 15, 146 (1997); Nature Biotech., 18, 1086 (2000); Proc. Natl. A cad. Acad. Sci. USA, 97, 722 (2000); Nucleic Acids Res., 33, e85 (2005); Metho ds Mol. Biol, 240, 207 (2004); Biotechnol. Genet.
  • cDNA is prepared from a tissue or cell of a human or non-human animal using a probe primer specific for the antibody molecule of interest in accordance with the “cDNA preparation method” described in 1. above. be able to.
  • an expression vector one that can replicate autonomously in the above non-human animal or can be integrated into a chromosome and contains a promoter at a position where DNA encoding the target antibody molecule can be transcribed is used. It is done.
  • expression vectors include, for example, pcDNAI, pcDM8 (commercially available from Funakoshi), PAGE107 [JP-A-3-22979; Cytotechnology, 3, 133, (199 0)], pAS3 -3 [JP-A-2-227075], pCDM8 [Nature, 329, 840, (1987)], pc DNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [Journal 'Ob' Biochemistry (J. Biochemistry), dish, 1307 (1987)], pAGE210, and the like.
  • Any promoter can be used as long as it can be expressed in animal cells.
  • a promoter of the cytomegalovirus (CMV) IE (immediate early) gene an early promoter of SV40, a retrovirus-less promoter. Examples include promoters, metamouthone promoters, heat shock promoters, SRa promoters, various yarn and fabric specific promoters, and the like.
  • CMV IE gene enhancer is a promoter. You may use with one.
  • any method for introducing the recombinant vector any method can be used as long as it introduces DNA into an embryonic stem cell or fertilized egg of an animal.
  • the electoral position method [Cytotechnology, 3, 133 (1990)]
  • calcium phosphate method [Japanese Patent Laid-Open No. 2-22707 5]
  • lipofusion method [Proceedings of the National Academy of Sciences, Proc. Natl. Acad. Sci. USA), 84, 7413 (1987)]
  • injection method Manipulating the Mouse Embryo A Laboratory Manual, Second Edition, Cold Spring Harbor or Laboratory Press (1994) Abbreviation
  • a method using a particle gun (gene gun) [Patent No. 2606856, Patent No.
  • DEAE-dextran method Biomanual series 4 single gene introduction and expression and analysis method (Yodosha) Takashi Yokota 'Arai Kenichi Ed. (1994)] virus vector method [Mupureating 'Mouse Embryo 2nd edition], and the like.
  • Examples of a method for obtaining a transformed individual producing an antibody molecule include a method using a prepared embryonic stem cell or fertilized egg into which a recombinant vector has been introduced.
  • embryonic stem cells prepared by introducing a recombinant vector When using embryonic stem cells prepared by introducing a recombinant vector, they are introduced into fertilized eggs according to the known injection chimera method or assembly chimera method, and the introduced fertilized eggs are introduced into the oviduct or uterus of pseudopregnant female mice.
  • a germline chimera is obtained by transplanting, the obtained germline chimera is mated, and an individual having a chromosome into which the introduced recombinant vector is inserted is selected from the pups born. It is possible to obtain transformed individuals to produce.
  • the fertilized egg introduced with the gene is transplanted into the oviduct or uterus of a pseudopregnant female mouse, and the introduced recombinant vector is introduced from the pup.
  • the introduced recombinant vector is introduced from the pup.
  • the target antibody gene is expressed in non-human animals such as rabbits, hidges, goats, pigs, horses, mice, rats, rabbits, monkeys, and rabbits that have been introduced with genes involved in sugar chain synthesis.
  • an antibody molecule to which a sugar or a sugar chain is added by the introduced gene can be obtained.
  • the transformed individual obtained as described above is bred according to a usual method, an antibody composition is produced and accumulated, and the antibody composition is collected from the animal individual to obtain the antibody composition. Can be manufactured.
  • an antibody composition is produced by breeding a non-human animal into which DNA encoding an antibody molecule is introduced, generating and accumulating the antibody composition in the animal, and collecting the antibody composition from the animal. It can be done.
  • Examples of the production / accumulation location in the animal include tissues and body fluids.
  • milk Japanese Patent Laid-Open No. 63-309192
  • eggs of the animal can be mentioned as preferable examples.
  • the promoter used in this case can be used as long as it can be expressed in animals.
  • ⁇ -casein promoter, ⁇ -casein promoter, 13-lactoglobulin promoter which are mammary cell-specific promoters.
  • suitable promoters include whey acid 'protein promoter.
  • a non-human animal introduced with DNA encoding an antibody molecule ⁇ ! Is raised cells that produce the target antibody molecule are taken out from the non-human animal, and the obtained production cells are cultured in a medium.
  • An antibody composition can also be produced by generating and accumulating an antibody composition in a product, and collecting and purifying the antibody composition from the culture.
  • Non-human animal forces can also acquire cells by using animal tissue culture, Modern Biology 1 'Series 23, Kyoritsu Shuppan, (1974), Cell Culture Manual, Kodansha Scientific, (1982), Use of Isolated Liver. ells and Kidney Tubules in Metabolic Studies, Elaevi er / North— Holland Publishing Co., New York-Amsterdam-Oxford, (1976), Techniques for Illustrative Animal Experiments, Protein Nucleic Acid Enzyme Volume No. 24, (1981), Mouse Lab manual, focusing on gene introduction and analysis, Tokyo Metropolitan Institute of Medical Science, Laboratory Animal Research Division, Springer 'Fairark Tokyo Co., Ltd. (1998), Mouse Lab Manual [2nd edition], There are known methods described in the post-genome era experimental methods, the Department of Experimental Animal Research, Tokyo Metropolitan Institute of Medical Science, Springer's Fairlark Tokyo Co., Ltd. (2003), and the like.
  • the obtained cells can be immortalized by performing continuous culture in a non-aggregated state aseptically for at least 2 months.
  • a cancer gene may be introduced to promote immortalization of the cells.
  • Oncogenes include, for example, SV40 large T antigen, Ras, Myc
  • V ⁇ is a known oncogene that is already known to promote immortalization when introduced into primary cultured cells such as Src. Immortalized cells can be efficiently obtained by incorporating these oncogenes into the appropriate expression vectors for animal cells described above and introducing them into the obtained cells.
  • a method for culturing the obtained production cells in a medium can be performed according to a conventional method used for culturing cells.
  • the scallop can be a medium obtained by adding fetal calf serum or the like to these mediums.
  • Cultivation is usually performed under conditions such as pH 6-8, 30-40 ° C, and the presence of 5% CO 1
  • antibiotics such as kanamycin and penicillin may be added to the medium as needed during the culture.
  • a method for producing an antibody composition there are a method of producing intracellularly, a method of secreting it extracellularly, or a method of producing it on the outer membrane, and the cells used for production and the antibody molecules produced are produced.
  • the method can be selected by changing the structure.
  • the antibody composition When the antibody composition is produced intracellularly or on the outer membrane, the method of Paulson et al. [Journal 'Ob. Biological' Chemistry (J. Biol. Chem.), 264, 17619 (1989)], Rowe et al. (Proc. Natl. Acad. Sci. USA), 86, 8227 (1989); Gene Development (Genes Develop.), 4 , 1288 (1990)], or by applying the method described in JP-A-05-336963, JP-A-06-823021, etc., the antibody can be actively secreted extracellularly.
  • DNA encoding an antibody and DNA encoding a signal peptide appropriate for expression of the antibody are inserted into an expression vector, and the expression vector is introduced into the antibody.
  • the target antibody molecule can be actively secreted out of the cell.
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • An antibody composition produced by a transgenic individual into which a gene encoding an antibody molecule has been introduced is, for example, when the antibody composition is expressed in a dissolved state in the cell, and after culturing, the cell is centrifuged. Collect and suspend in an aqueous buffer, then crush the cells with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dynomill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Tylaminoethyl (DEAE) —Sepharose, DIAION HPA-75 (Mitsubishi Chemical)
  • Anion-exchange chromatography using resin such as), cation-exchange chromatography using resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose and ferrule sepharose
  • An antibody composition using a method such as a hydrophobic chromatography method, a gel filtration method using a molecular sieve, a affinity chromatography method, a chromatofocusing method, or an electrophoresis method such as isoelectric focusing alone or in combination.
  • the purified sample can be obtained.
  • the cell When the antibody composition is expressed in the form of an insoluble substance in the cell, the cell is similarly collected, crushed, and centrifuged to obtain an insoluble antibody composition as a precipitate fraction. Collect.
  • the recovered insoluble body of the antibody composition is solubilized with a protein denaturant. By diluting or dialyzing the solubilized solution, the antibody is returned to a normal three-dimensional structure, and then a purified preparation 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 recovered in the culture supernatant. That is, the soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and the antibody composition is obtained from the soluble fraction by using the same isolation and purification method as described above. The purified sample can be obtained.
  • Examples of the antibody composition thus obtained include, for example, a human antibody composition, a chimeric antibody composition, a humanized antibody composition, an antibody fragment composition having an Fc region of those antibodies, and an Fc region of these antibodies. And a fusion protein composition with other proteins.
  • the purified glycoprotein composition is labeled with a compound such as a radioisotope, and the binding reaction with the labeled glycoprotein composition receptor or interacting protein is enhanced.
  • a compound such as a radioisotope
  • protein-protein interaction can be measured using various devices such as Biacore's BIAcore series (J. Immnunol. Methods, 145, 229 (1991)). Intermolecular interaction experiment method, Yodosha (1996)).
  • the binding activity to the antigen and the binding activity to the antigen-positive cultured cell line are determined by ELISA and fluorescent antibody.
  • Method [Cancer Immunol. Immunother., 36, 373 (1993)] and the like. Cytotoxic activity against antigen-positive cell lines can be evaluated by measuring CDC activity, ADCC activity, etc. [Cancer Immunol. Immunother., 36, 373 (1993)].
  • sugar chain structure analysis is based on the structure of normal glycoproteins. This can be done according to the structure analysis.
  • sugar chains bound to IgG molecules are composed of neutral sugars such as galatatose, mannose, and fucose, amino sugars such as N-acetylyldarcosamine, and acidic sugars such as sialic acid.
  • Sugar chain structure analysis can be performed using sugar composition analysis, two-dimensional sugar chain mapping, and the like.
  • composition analysis of the sugar chain of the antibody molecule can be analyzed by first conducting acid hydrolysis of the sugar chain with trifluoroacetic acid or the like to release neutral sugars or amino sugars, and measuring the composition ratio.
  • a specific method includes a method using a sugar composition analyzer (BioLC) manufactured by Dionex.
  • BioL is HPAE and sugar composition by high performance amon- exchange chromatography-pulsed amperometric detection (J. Liq. Chromatogr.), 6,1577 (1983) It is a device that analyzes.
  • the sugar composition ratio can also be analyzed using the fluorescent labeling method with 2-aminoviridine. Specifically, a sample hydrolyzed according to a known method [Agricultural 'and' biological 'chemistry (Agruc. Biol. Chem.), 55 (1), 283-284 (1991)] An aminoviridylation reaction is performed, fluorescent labeling is performed, and the labeled sample is analyzed using HPLC, and the composition ratio can be calculated.
  • the structure analysis of glycans in antibody molecules is based on the two-dimensional glycan mapping method [Analytical 'Biochem. (Anal. Biochem.),, 73 (1988), Biochemical Experimental Methods 23-Glycoprotein Glycan Research Methods Publication Center) Reiko Takahashi (1989)].
  • the 2D glycan mapping method for example, the retention time or elution position of reversed-phase chromatography glycans is plotted on the X axis, and the retention time or elution position of glycans by normal phase chromatography is plotted on the Y axis.
  • the sugar chain structure is estimated by comparing with analysis data of known sugar chains.
  • the antibody is hydrazine-degraded to release the sugar chain from the antibody, and 2-aminoviridine (hereinafter abbreviated as PA) is used to fluorescently label the sugar chain [Journal of Biochemistry (j. Biochem.), 95, 197 (1984)], followed by gel filtration of the sugar chain with excess PA reagent etc. followed by reverse phase chromatography. Perform normal phase chromatography on each peak of the separated sugar chain. These results are plotted on a two-dimensional glycan map and described in the glycan standard (TaKaRa), literature [Anal. Biochem., 171, 73 (1988)].
  • the sugar chain structure can be estimated by comparing spots with known sugar chains.
  • the structure can be estimated and confirmed according to the two-dimensional sugar chain mapping method.
  • glycoprotein composition produced using the non-human animal of the present invention or its progeny is subjected to fucose modification. For example, increased affinity with the receptor, prolonged blood half-life, improved tissue distribution after blood administration, or increased interaction with other proteins. As a result, effects such as improved pharmacological activity can be expected, and high physiological activity is exhibited.
  • the antibody composition may have a high effector function, ie, antibody-dependent cellular cytotoxicity (ADCC activity).
  • ADCC activity antibody-dependent cellular cytotoxicity
  • These highly bioactive sucrose protein compositions, or antibody compositions having high ADCC activity are, for example, cancer, inflammatory diseases, autoimmune diseases, immune diseases such as allergies, cardiovascular diseases, viruses or It is useful in the prevention and treatment of various diseases caused by bacterial infections.
  • cancer that is, malignant tumor
  • existing anticancer agents are characterized by inhibiting the growth of cancer cells.
  • an antibody having high ADCC activity exhibits a cytocidal effect on cancer cells and can treat cancer, and is therefore a more effective therapeutic agent than existing anticancer agents.
  • the anti-tumor effects of single administration of antibody drugs are often inadequate at present, so combination therapy with chemotherapy has been carried out (Science ence), 280, 1197, 1998]. Therefore, if a strong antitumor effect is observed by single administration of the antibody of the present invention, the dose of the chemotherapeutic agent used in combination can be reduced, leading to reduction of side effects.
  • cardiovascular diseases examples include arteriosclerosis.
  • a balloon catheter is currently used for the treatment of arteriosclerosis, but it causes arteriosclerosis at the restenosis site that occurs after treatment! / Highly proliferating cells ⁇
  • Antibody composition with ADCC activity Cardiovascular diseases can be prevented and treated by controlling with the use of substances.
  • Antibodies that recognize tumor-related antigens antibodies that recognize antigens related to allergy or inflammation, antibodies that recognize antigens related to cardiovascular diseases, antibodies that recognize antigens related to autoimmune diseases, or viruses
  • Specific examples of antibodies that recognize antigens associated with bacterial infection are described below.
  • Antibodies that recognize tumor-associated antigens include anti-CA125 antibody (Immunology Today, 21, 03-410, 2000), anti-17-1A antibody (Immunology Today, 21, 403-410, 2000), anti-integrin. av jS 3 antibody (Immunology Today, 21, 403-410, 2000), anti-CD33 antibody (Immunology Today, 21, 403-410, 2000), anti-CD22 antibody (Immunology Today, 21, 403-410, 2000) , Anti-HLA antibody (Immunology Today, 21, 403-410, 2000), anti-HLA-DR antibody (Immunology Today, 21, 403-410, 2000), anti-CD20 antibody (Immunology Today, 21, 403-410, 2000) ), Anti-CD 19 antibody (Immunology Today, 21, 403-410, 2000), anti-EGF receptor antibody (Immunology Today, 21, 403-410, 2000), anti-CD 10 antibody (American Journal of Clinical Pathology, 113, 374-382, 2000; Proc
  • Anti-HM1.24 antibody (Molecular Immunol, 36, 387-395, 1999), anti-parathyroid hormone related protein (PTHrP) antibody (Cancer, 88, 2909-2911, 2000), anti-FG F8 antibody (Proc. Natl. Acad. Sci. USA, 86. 9911-9915, 1989) Increased anti-basic fibroblasts Growth factor antibody, anti-FGF8 receptor antibody (J. Biol. Chem., 265, 16455-16463, 1990), anti-basic fibroblast growth factor receptor antibody, anti-insulin-like growth factor antibody 0. Neurosci. R es., 40, 647-659, 1995), anti-insulin-like growth factor receptor antibody (J.
  • Antibodies that recognize antigens related to allergy or inflammation include anti-IgE antibodies (Immunology Today, 21, 403-410, 2000), anti-CD23 antibodies (Immunology Today, 21, 403-410, 2000), Anti-CD 11a antibody (Immunology Today, 21, 403-410, 2000), anti-CRTH2 antibody (J. Immunol, 162, 1278-1286, 1999), anti-CCR8 antibody (W099 / 25734), anti-CCR 3 antibody (US6207155 ), Anti-interleukin 6 antibody (Immunol.
  • Anti-GpIIbZlIIa antibody J. Im ⁇ nol., 152, 2968-2976, 1994
  • antiplatelet-derived growth factor antibody Science, 253, 1129-
  • Antiplatelet-derived growth factor antibody is recognized as antibodies that recognize antigens related to cardiovascular diseases. 113 2, 1991
  • antiplatelet-derived growth factor receptor antibody J. Biol. Chem., 272, 17400-17404, 1997) or anticoagulation factor antibody (Circulation, 101, 1158-1164, 2000) It is done.
  • Anti-self DNA antibodies include antibodies that recognize antigens associated with autoimmune diseases such as psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, and multiple sclerosis.
  • anti-CD 11a antibody (Immunology Today, 21, 403-410, 2000), anti-ICAM3 antibody (Immunology Today, 21, 403-410, 2000), anti-C D80 antibody (Immunology Today, 21, 403-410, 2000), anti-CD2 antibody (Immunology Today, 21, 403-410, 2000), anti-CD3 antibody (Immunology Today, 21, 403-410, 2000), anti-C D4 antibody (Immunology Today, 21, 403-410, 2000), anti-integrin a 4 j8 7 antibody (Immunology Today, 21, 403-410, 2000), anti-CD40L antibody (Immunology Today, 21, 403-410, 2000) ) And anti-IL 2 receptor antibodies (Immunology Today, 21, 403-410, 2000).
  • Anti-gpl20 antibody (Structure, 8, 385-395, 2000), anti-CD4 antibody (J. Rheumatology, 25, 2065-2076, 1 998) ), Anti-CCR4 antibody or anti-verotoxin antibody (J. Clin. Microbiol, 37, 396-399, 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, Dainippon Pharmaceutical Co., Ltd., R & D SYSTEMS, PharMingen. , Cosmo Bio, Funakoshi Co., Ltd.
  • a drug containing the glycoprotein composition of the present invention can be administered alone as a therapeutic agent. Usually mixed with one or more pharmacologically acceptable carriers. It is desirable to provide it as a pharmaceutical preparation produced by any method well known in the technical field of pharmaceutics.
  • the route of administration can be oral administration where it is desirable to use the most effective treatment, or parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous.
  • parenteral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular and intravenous.
  • intravenous administration can be preferably mentioned.
  • administration forms include sprays, capsules, tablets, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
  • Suitable formulations for oral administration include emulsions, syrups, capsules, tablets, powders, granules and the like.
  • Liquid preparations such as emulsions and syrups include sugars such as water, sucrose, sorbitol and fructose, darikols such as polyethylene glycol and propylene glycol, oils such as sesame oil, olive oil and soybean oil, p-hydroxy Preservatives such as benzoate esters It can be produced using flavors such as single flavor and peppermint as additives.
  • Capsules, tablets, powders, granules and the like are excipients such as lactose, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc. It can be produced using a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • a binder such as an agent, polybulal alcohol, hydroxypropylcellulose, gelatin, a surfactant such as a fatty acid ester, a plasticizer such as glycerin, and the like as additives.
  • Suitable preparations for parenteral administration include injections, suppositories, sprays and the like.
  • the injection is prepared using a carrier such as a salt solution, a glucose solution, or a mixture of both.
  • a powder injection can be prepared by freeze-drying a glycoprotein composition according to a conventional method and adding sodium chloride thereto.
  • Suppositories are prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
  • the propellant does not irritate the glycoprotein composition itself or the recipient's oral cavity and airway mucous membrane, and the carrier disperses the glycoprotein composition as fine particles to facilitate absorption. It is prepared using.
  • the carrier include lactose and glycerin.
  • preparations such as aerosols and dry powders are possible.
  • the components exemplified as additives for oral agents can also be added.
  • the dose or frequency of administration varies depending on the intended therapeutic effect, administration method, treatment period, age, body weight, etc.
  • the normal adult dose is 10 / z g / kg to 20 mg / kg per day.
  • in vitro experiments include a CDC activity measurement method, an ADCC activity measurement method and the like
  • in vivo experiments include methods for examining the antitumor effect of glycoprotein compositions on various tumor cells. And anti-tumor experiments using tumor systems in laboratory animals such as mice.
  • Plasmid mcKOgE2-1 was constructed according to the following procedure (Fig. 1).
  • Plasmid mcgE2-0-XbaI (2.0 mg) was dissolved in NEBuffer2 (New England Biolabs) 35 ⁇ 1 containing 100 ⁇ g / ml BSA (New England Biolabs), and the restriction enzyme HindIII (New England Biolabs) 10 The digestion reaction was performed at 37 ° C for 2.5 hours with 10 units and 10 units of XmnI (New England Biolabs).
  • mcgE2-0-XbaI is an Xbal-Xbal fragment (about 3.0 Kb) encoding the exon and upstream intron region of the Fut8 translation initiation codon obtained in Reference Example 1, and pBluescriptll KS (+) (Strategene) It is a plasmid inserted into After the reaction, the digestion reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 3.5 Kb was purified with GENEC LEAN Spin Kit (BIO 101) and eluted with water 201 (hereinafter referred to as agarose gel). This method was used to purify force DNA fragments).
  • plasmid pBlueScriptll SK (+) (Strategene) was dissolved in 35 ⁇ 1 of NEBufferl (New England Biolabs) containing 100 ⁇ g / ml BSA (New England Biolabs), and the restriction enzyme was used. Digestion reaction was carried out at 37 ° C for 2.5 hours with 10 units of SacI (New England Biolabs) and 10 units of XmnI (New England Biolabs). The digestion reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.9 Kb was purified.
  • Xmnl-Hindlll fragment (about 3.5 Kb) 1.0 1, derived from the plasmid mcgE2-0-Xbal obtained above Hindlll-Sad fragment from plasmid mcgE2- 0- Xbal (approximately 330 bp) 1.0 1, Sad-Xmnl fragment from plasmid pBlueS criptll SK (+) (approximately 1.9 Kb) 0.5 ⁇ 1, water 2.5 ⁇ 1, Ligation High ( Toyobo Co., Ltd.) 5.0 1 was mixed and the fragments were ligated by reacting at 16 ° C for 1 hour.
  • the reaction solution is used to transform Escherichia coli DH5a strain, and a method known from the resulting ampicillin resistant clone [Nucleic Acids Research, 7, 1513 (1979)] Each plasmid DNA was isolated according to this method). This plasmid is hereinafter referred to as mcKOgE2-l.
  • Plasmid pKOScramber901 / ARI was constructed according to the following procedure (Fig. 2).
  • plasmid pKOScramberV901 (Lexicon Genetics) was dissolved in 35 ⁇ 1 of NEBuffer for EcoRI (New England Biolabs) and digested with 10 units of restriction enzyme EcoRI (New England Biolabs). The digestion reaction solution was subjected to ethanol precipitation, and then the vector digestion end was smoothed with Blunting High (Toyobo) in 101 reaction system. The smoothing reaction solution 1.01, water 4.01, and Ligation High (Toyobo Co., Ltd.) 5.01 were mixed, and the fragments were ligated by reacting at 16 ° C for 1 hour.
  • Escherichia coli DH5a strain was transformed with the reaction solution, and plasmid DNAs were isolated from the resulting ampicillin resistant clones according to a known method.
  • This plasmid is hereinafter referred to as pKOScramber901 / ARI.
  • Plasmid mcKOgE2-2 was constructed according to the following procedure (Fig. 3).
  • Xba to BssHII fragment (about 2.5 Kb) 4.5 1 derived from plasmid mcKOgE2-l obtained above, Hindlll-Ascl fragment (about 1.9 Kb) 0.5 ⁇ 1, derived from plasmid pKOScramber901 / ARI, about 1.5 Kb, Ligation Hi gh (Toyobo) 5.0) was mixed and the fragments were ligated by reacting at 16 ° C for 30 minutes. Escherichia coli DH5a strain was transformed with the reaction solution, and plasmid DNA was isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as mcKOg E2-2.
  • Plasmid mcKOgE2-3 was constructed according to the following procedure (Fig. 4).
  • plasmid pKOSelectDT (Lexicon Genetics) was dissolved in 35 ⁇ 1 of NEBuffer4 (New England Biolabs), and 10 units of restriction enzyme Rsrll (New England Biolabs) were collected. A half hour digestion reaction was performed. The digestion reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 1.2 Kb was purified.
  • Plasmid mcKOgE2-4 was constructed by the following procedure (FIG. 5).
  • oligonucleotides with a restriction enzyme EcoRI reaction site at the 5 'end and a restriction enzyme Hindlll reaction site at the 3' end via the ⁇ site and the Fut8 intron region (SEQ ID NO: 11 and SEQ ID NO: 12) was synthesized.
  • Each synthetic oligonucleotide was dissolved in 20 1 water so as to be 5 / z mol / 1 and heated at 100 ° C. for 3 minutes. After cooling at room temperature, have rows phosphorus Sani ⁇ oligonucleotide end 25 1 of the reaction system by using a MEGALABEL T M (Takara Bio Inc.) was diluted 10-fold with water.
  • Plasmid mcKOgE2-5 was constructed by the following procedure (Fig. 6).
  • mcgE2-0-SacI is the position of the Fut8 translation initiation codon obtained in Reference Example 1. This is a plasmid in which a Sacl-Sacl fragment (about 6.3 Kb) coding for Rxon and downstream intron region is inserted into pBluescript KS (+) (Strategene). After the reaction, the digestion reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 4.3 Kb was purified.
  • Pstl-Pstl fragment (approx. 4.3 Kb) derived from plasmid mcgE2-0-Sad obtained above 0.5 ⁇ 1, water 4.5 ⁇ Ligation High (Toyobo Co., Ltd.) 5.0 1 were mixed and mixed at 16 ° C for 30 minutes The pieces were connected by reaction. Escherichia coli DH5a strain was transformed with the reaction solution, and plasmid DNA was isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as mcKOgE2-5.
  • Plasmid mcKOgE2-6 was constructed by the following procedure (Fig. 7).
  • an oligonucleotide having a restriction enzyme Nhel reaction site at both ends via a unit in which the FRT site, restriction enzyme Ascl reaction site, FRT site, and ⁇ site are linked in the forward direction (SEQ ID NO: 13 and SEQ ID NO: 14) was synthesized.
  • Each synthetic oligonucleotide was dissolved in 20 1 water at a concentration of 5 mol / 1 and heated at 100 ° C. for 3 minutes. After allowing to cool at room temperature, phosphorylation of the oligonucleotide ends was performed in a 25 ⁇ 1 reaction system using MEGALA BEL TM (Takara Bio Inc.) and diluted 10-fold with water.
  • Plasmid mcKOgE2-7 was constructed according to the following procedure (FIG. 8).
  • Plasmid mcKOgE2-9.1 was constructed according to the following procedure (Fig. 9).
  • Example 5 of this Example 1.0 ⁇ g of the plasmid mcKOgE2-4 4 obtained in Example 5 of this Example was added to NEBuffer for Sal I (New England Biolabs) containing 100 ⁇ g / ml BSA (New England Biolabs) 35 ⁇ m. Dissolve in 1 and add 10 units of restriction enzyme Sal I (New England Biolabs) at 37 ° C for 2.5 hours. The reaction was carried out.
  • reaction mixture After subjecting the reaction mixture to ethanol precipitation, it is dissolved in NEBufferl (New England Biolabs) 35 ⁇ 1 containing 100 ⁇ g / ml BSA (New England Biolabs) and the restriction enzyme Sac I (New England Biolabs) The digestion reaction was performed at 37 ° C for 2 hours with 10 units added. The reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 5.7 Kb was purified.
  • NEBufferl New England Biolabs 35 ⁇ 1 containing 100 ⁇ g / ml BSA (New England Biolabs) and the restriction enzyme Sac I (New England Biolabs)
  • Sac I New England Biolabs
  • Plasmid pKOSelectNeo / ASac was constructed by the following procedure (FIG. 10).
  • Plasmid pKOSelectNeo (Lexicon Genetics) 1.0 ⁇ g was dissolved in NEBufferl (New England Biolabs) 35 ⁇ 1 containing 100 ⁇ g / ml BSA (New England Biolabs), and the restriction enzyme Sac I ( New England Biolabs) Digested with 10 units. After subjecting the digestion reaction solution to ethanol precipitation, the vector digestion end was smoothed in a reaction system of 101 by Blunting High (Toyobo Co., Ltd.). The smoothing reaction solution 1.0 1, water 4.0 ⁇ 1, and Ligation High (Toyobo Co., Ltd.) 5.0 ⁇ 1 were mixed, and the fragments were ligated by reacting at 16 ° C.
  • Escherichia coli DH5a strain was transformed using the reaction solution, and plasmid DNAs were isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pKOSelectNeo / ASac.
  • Plasmid pmcKOFUT8Neo was constructed according to the following procedure (FIG. 11).
  • DNA end was dephosphorylated by adding ase (Takara Bio) 1.0 1 and reacting at 65 ° C for 30 minutes.
  • the reaction solution was subjected to 0.8% (w / v) agarose gel electrophoresis, and a DNA fragment of about 11.2 Kb was purified.
  • Asc I-Asc I fragment (about 1.5 Kb) derived from plasmid pKOSelectNeo / ASac obtained above (about 1.5 Kb) 1.0 ⁇ 1, Asc I-Asc I fragment (about 11.2 Kb) 1.0 ⁇ 1, derived from plasmid mcKOgE2-9.1, water
  • the fragments were joined by mixing 3.0 ⁇ 1 and Ligation High (Toyobo Co., Ltd.) 5.0 1 and reacting at 16 ° C. for 1.5 hours.
  • Escherichia coli Stbl2 strain was transformed using the reaction solution, and plasmid DNA was isolated from the resulting ampicillin resistant clones according to a known method. This plasmid is hereinafter referred to as pmcKOFUT8Neo.
  • Mouse embryonic stem cells used for homologous recombination were maintained and cultured using mouse primary fibroblasts (EMFI cells) prepared according to the following description as feeders.
  • EMFI cells mouse primary fibroblasts
  • mitomycin-treated EMFI cells were treated with FM medium [10% ushi fetal serum (Invitrogen), 55 ⁇ mol / 1 mercaptoethanol (Invitrogen), 1 mmol / 1 MEM sodium pyruvate (Invitrogen), 0.1 mmol / 1 MEM non-essential amino acids (Invitrogen), 3 mmol / 1 adenosine (SIGMA), 3 mmol / 1 guanosine (SIGMA), 3 mmol / 1 cytidine (SIGMA), 3 mmol / 1 uridine ( SIGMA) Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 1 mmol / 1 thymidine (SIGMA), 2 mmol / 1 L-glutamine (Invitrogen), 100 units / ml penicillin and 100 ⁇ g / ml streptomycin (Invitrogen) )]
  • FM medium 10%
  • Mouse embryonic stem cells D3 (provided by Professor Akira Muramatsu, Faculty of Medicine, Nagoya University, and maintained at the Osaka University Genetic Information Experiment Center) were cultured in ESM medium [20% Usushi Fetal Serum (Invitrogen), 55 ⁇ mol / 1 ⁇ -mercaptoethanol (Invitrogen), 1 mmol / 1 MEM sodium pyruvate (Invitrogen), 0.1 mmol / 1 MEM non-essential amino acid (Invitrogen), 3 mmol / l adenosine (SIGMA), 3 mmol / l guanosine ( SIGMA), 3 mmol / 1 cytidine (SIGMA), 3 mmol / 1 uridine (SIGMA), 1 mmol / 1 thymidine (SIGMA), 2 mmol / 1 L-glutamine (Invitrogen), 100 units / Dulbecco's Modified Eagle Medium (DMEM; In
  • D3 cells were subcultured to three feeder plates prepared as described above.
  • Targeting vector plasmid obtained in Example 1 into D3 cells was performed according to the following procedure according to the eletroporation method [Cytotechnology, 3, 133 (1990)].
  • 20 ⁇ g of the targeting vector plasmid was linearly digested with the restriction enzyme Not I, followed by phenol / chloroform extraction and ethanol precipitation to give a 1 g / 1 solution.
  • the culture supernatant was removed from D3 cells 48 hours after subculture and replaced with fresh ESM medium. After confirming that D3 cells reached 70% confluence, the suspension was suspended in PBS buffer (Invitrogen) to give 1 ⁇ 10 7 cells / ml.
  • Cell suspension 800 1 (8.0 X 10 6 cells) is mixed with 20 g of the above linear plasmid and then cell DN Transfer the entire volume of the mixture A to Gene Pulser Cuvette (distance between electrodes 4 mm) (BI ⁇ -RAD), and use a cell fusion device Gene Pulser (BIO-RAD) with a pulse voltage of 250 V and an electric capacity of 500 ⁇ F. Gene transfer was performed under conditions. After the introduction, the cell suspension was suspended in 40 ml of ESM medium, and seeded on 3 feeder plates prepared using 10 cm diameter dishes and 2 feeder plates prepared using 6 cm diameter dishes. 5% CO
  • the 96 clones obtained in Section 3 of this example were diagnosed for homologous recombination by genomic PCR according to the following procedure.
  • genomic DNA of each clone was determined according to a known method [Nucleic Acids Research, 3, 2303, (1976)] from the replica plate prepared in Section 3 of this Example. Each was prepared and dissolved in TE buffer (pH 8.0) [10 mmol / l Tris-HC1, lmmol / 1 EDTA].
  • TE buffer pH 8.0
  • a forward primer SEQ ID NO: 15 and SEQ ID NO: 16
  • Reverse primers SEQ ID NO: 17 and SEQ ID NO: 18
  • PCR polymerase chain reaction
  • mice 1. Obtain chimera mice using embryonic stem cells with one copy of the Fut8 allele recombined. Two clones of embryonic stem cells that have been recombined with one Fut8 allele, established in Example 2, were used as a guide 'two' Techniques 'in'. Microinjection into the blastocyst space obtained from female C57BL / 6 strain mice as described in Mouse's Development [Guide to Technuques in Mouse Development, Methods in Enzymology, Volume 225, Academic Press (1993)] It was. After the injection, the mice were transplanted and implanted into the pupae of MCH female mice pseudo-pregnant.
  • the chimera individuals obtained in section 1 of this example were raised to 8 weeks of age, and then mated with sex-matured C57BL / 6 females to obtain offspring.
  • a genomic DNA was prepared from the tail of an individual having brown coat among the offsprings according to a known method [Nucleic Acids Research, 3, 2303, (197 6)]. 2 Southern plot analysis was performed according to the method described in Section 5.
  • Genomic DNA of the heterozygote was digested with the restriction enzyme Sac I to produce a wild-type Fut8 allele-specific fragment of approximately 6.3 Kb and an allele-specific fragment of approximately 8.0 Kb that had undergone homologous recombination. It occurs at a volume ratio of 1: 1 ( Figure 12).
  • Figure 12 As a result of Southern plot analysis, it was confirmed that a heterozygote satisfying the above criteria was obtained from a chimeric individual derived from an embryonic stem cell that was found to contribute to the germline in paragraph 1 of this Example. .
  • Homozygous genomic DNA yields only about 8.0 Kb of allele-specific fragments that have undergone homologous thread replacement by restriction enzyme Sac I treatment (Fig. 12). As a result of Southern plot analysis, it was confirmed that homozygotes satisfying the above criteria were included in the litter.
  • C.129P2-Cdl9 tml (ere) n / J is a mouse in which the translation region of the CD19 gene on the chromosome is replaced with the translation region of the Cre recombinase gene.
  • genomic DNA was prepared from the obtained offspring tail according to a known method [Nucleic Acids Research, 3, 2303, (1976)].
  • a forward primer (SEQ ID NO: 19) and reverse primer (SEQ ID NO: 20) that bind to the Cre recombinase gene a forward primer (SEQ ID NO: 21) and a reverse primer (SEQ ID NO: 22) that bind to the CD19 gene, Were synthesized respectively.
  • This genomic PCR produces a specific amplified fragment of 100 bp from the Cre recombinase gene and a specific amplified fragment of 477 bp from the CD 19 gene.
  • a baby pup was observed in which Cre-recombinase gene-specific amplification was observed.
  • This offspring is an individual that has the Cre recombinase gene on its chromosome and is heterozygous for the Fut8 gene.
  • PCR was performed using DNA polymerase Ex Taq (Takara Shuzo Co., Ltd.) with the above genomic DNA solution in a bowl shape and primers shown in IJ Nos. 19, 20, 21, and 22. PCR was performed under conditions of 37 cycles of heating at 94 ° C for 2 minutes, followed by a reaction consisting of 94 ° C for 20 seconds, 64 ° C for 30 seconds, and 72 ° C for 50 seconds.
  • This genomic PCR generates a specific amplified fragment of 100 bp from the Cre recombinase gene and a specific amplified fragment of 477 bp from the CD 19 gene.
  • agarose gel electrophoresis of the PCR reaction solution we found offspring that showed specific amplification of the Cre recombinase gene.
  • genomic clone was digested with various restriction enzymes, and then the molecular clone 2nd edition [Molecul] using the above porcine FUT8 cDNA partial fragment 412bp as a probe. Southern blotting was performed according to a known method described in ar Cloning, A Laboratory Manual, 2nd Ed. (1989)].
  • ⁇ -1,6-fucose modifying enzyme the activity of ⁇ -1,6-fucose modifying enzyme, non-human animals whose genome has been modified so as to be reduced or deleted in a tissue-specific manner, their progeny, methods for producing the same, and uses thereof A method is provided.
  • SEQ ID NO: 11 Description of artificial sequence: synthetic DNA
  • SEQ ID NO: 12 Description of artificial sequence: synthetic DNA
  • SEQ ID NO: 17 Description of artificial sequence: synthetic DNA

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Abstract

La présente invention concerne un animal non humain qui présente une réduction ou un défaut spécifique au tissu lors de l'activité d'une enzyme impliquée dans une modification de la chaîne de sucre. Selon l'invention, la modification de la chaîne de sucre se caractérise par la formation d'une liaison α entre la position 6 de la N-acétylglycosamine sur l'extrémité réductrice d'une chaîne de sucre composite liée au N-glycoside et la position 1 du fucose. L'invention porte aussi sur un procédé de production de l'animal non humain. En outre, l'invention a trait à l'utilisation de cet animal non humain.
PCT/JP2007/052873 2006-02-17 2007-02-16 Animal non humain possedant un genome modifie ou sa progeniture WO2007094462A1 (fr)

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Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
BRANDA C.S. AND DYMECKI S.M.: "Talking about a revolution: The impact of site-specific recombinationases on genetic analyses in mice", DEV. CELL, vol. 6, January 2004 (2004-01-01), pages 7 - 28, XP002994211 *
DATABASE GENBANK [online] HAYASHI H. ET AL.: "Molecular cloning of mouse alpha-1,6-fucosyltransferase and expression of its mRNA in the developing cerebrum", XP003016657, accession no. EMBL Database accession no. (AB025198) *
DE JASPER B. ET AL.: "Transgenic mice with hematopoietic and lymphoid specific expression of Cre", EUR. J. IMMUNOL., vol. 33, no. 2, February 2003 (2003-02-01), pages 314 - 325, XP003016658 *
DNA SEQ., vol. 11, no. 1-2, 2000, pages 91 - 96 *
IKEGAMI T. ET AL.: "Hai Sen'isho Hassho Yoin ni Okeru Insulin-yo Juyotai Signal", BINMANSEI HAI SHIKKAN KENKYUHAN, HEISEI 13 NENDO KENKYU HOKOKUSHO, 31 March 2002 (2002-03-31), pages 168 - 171, XP003016662 *
RAO M.K. ET AL.: "A highly active homeobox gene promoter regulated by Ets and Sp1 family members in normal granulosa cells and diverse tumor cell types", J. BIOL. CHEM., vol. 277, no. 29, 19 July 2002 (2002-07-19), pages 26036 - 26045, XP003016663 *
SATO M. ET AL.: "Kotai Iyaku to Tosa no Yakuwari", vol. 30, no. 12, 25 November 2004 (2004-11-25), pages 35 - 39, XP003016659 *
SELBERT S. ET AL.: "Efficient BLG-Cre mediated gene deletion in the mammary gland", TRANSGENIC RES., vol. 7, September 1998 (1998-09-01), pages 387 - 396, XP003016660 *
SHIMIZU T.: "No Sanka Stress Shogai Chikuseki Mouse o Mochiita No Roka Kiko no Kaiseki", LIFE SCIENCE FOUNDATION OF JAPAN NENPO, HEISEI 16 NENBAN, 1 March 2005 (2005-03-01), pages 6 - 8, XP003016661 *
UOZUMI N. ET AL.: "Purification and cDNA cloning of porcine brain GDP-L-Fuc:N-Acetyl-beta-D-GLucosaminide alpha1 6-Fucosyltransferase", J. BIOL. CHEM., vol. 271, no. 44, 1 November 1996 (1996-11-01), pages 27810 - 27817, XP003016656 *
YANAGIDANI S. ET AL.: "Purification and cDNA cloning of GDP-L-Fuc:N-Acetyl-beta-D-Glucosaminide: alpha1-6-Fucosyltransferase(alpha1-6FucT) from human gastric cancer MKN45 cells", J. BIOCHEM., vol. 121, no. 3, March 1997 (1997-03-01), pages 626 - 632, XP009003760 *

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